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Moseley, R. , Waddington, R.J. & Embery, G. Degradation of glycosaminoglycans by reactive oxygen species derived from stimulated polymorphonuclear leukocytes. Biochim. Biophys. Acta 1362, 221-231
Abstract
The effect of reactive oxygen species (ROS), generated by in vitro stimulation of isolated PMN upon the main GAG components of mineralised and non-mineralised connective tissues was investigated. PMN were isolated from whole blood and the production of the ROS superoxide (O2.-) and hydroxyl radicals (.OH) was stimulated by the addition of phorbol myristyl acetate (PMA) and PMA/FeCl3-EDTA chelate respectively and their production assessed over a 24 h period. The glycosaminoglycans (GAG), hyaluronan, chondroitin 4-sulphate and dermatan sulphate, were exposed to the ROS fluxes, incubated at 37 degrees C for 1 h and 24 h. GAG fragmentation was examined by gel exclusion chromatography and modification to hexuronic acid and hexosamine residues determined. Stimulation of PMN with PMA resulted in a burst of O2.- production for 1 h, which was sustained at a reduced level for 24 h. Fragmentation of GAG was observed for all GAG examined. Modification to the GAG was evident, with hyaluronan being more susceptible to loss of GAG residues than sulphated GAG. Modification of sugar residues increased with the incubation time and loss of the hexuronic acid residues was greater than loss of hexosamine residues. Addition of FeCl3-EDTA chelate, which led to the generation of .OH and was sustained over the 24 h period, demonstrated similar trends of GAG modification although increased degradation and loss of hexosamine and hexuronic acid were observed. GAG chains are constituents of PGs and their modification is likely to affect the function of these macromolecules and be of importance in considering the pathogenesis of inflammatory diseases, including periodontal diseases.
... Moreover, oxidative stress is an accepted component of aging and senescence [6,17,18]. The pathological outcomes of oxidative stress are attributed to the ROS-mediated modification of vital macromolecules, including nucleosides, lipids, and glycosaminoglycans (GAGs) in organisms [10,[19][20][21][22][23][24][25]. ...
... In this respect, HA is particularly susceptible since it is non-sulfated and contains β-linkages between the repeating units of β-1,4-d-glucuronic acid and β-1,3-N-acetyl-d-glucosamine [44]. Additionally, other GAGs have some resistance to ROS due to the presence of a covalently bound core protein at the reducing end, which is not the case with HA [20][21][22][23][24][25]45]. It has been established that HA is highly susceptible to be oxidized by ROS, resulting in the fragmentation of the HA chain [21][22][23][24][25][46][47][48]. ...
... Additionally, other GAGs have some resistance to ROS due to the presence of a covalently bound core protein at the reducing end, which is not the case with HA [20][21][22][23][24][25]45]. It has been established that HA is highly susceptible to be oxidized by ROS, resulting in the fragmentation of the HA chain [21][22][23][24][25][46][47][48]. Studies have also demonstrated that different ROS yield distinct HA products after fragmentation [23,46,49]. ...
A buildup of reactive oxygen species (ROS) occurs in virtually all pathological conditions. Hyaluronan (HA) is a major extracellular matrix component and is susceptible to oxidation by reactive oxygen species (ROS), yet the precise chemical structures of oxidized HA products (oxHA) and their physiological properties remain largely unknown. This study characterized the molecular weight (MW), structures, and physiological properties of oxHA. For this, high-molecular-weight HA (HMWHA) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) or hypochlorous acid (HOCl). ROS lead to the fragmentation of HA, with the oxHA products produced by HOCl exhibiting an altered chemical structure while those produced by H2O2 do not. HMWHA promotes the viability of human corneal epithelial cells (hTCEpi), while low MWHA (LMWHA), ultra-LMWHA (ULMWHA), and most forms of oxHA do not. HMWHA and LMWHA promote hTCEpi proliferation, while ULMWHA and all forms of oxHA do not. LMWHA and some forms of oxHA promote hTCEpi migration, while HMWHA does not. Finally, all native forms of HA and oxHA produced by HOCl promote in vivo corneal wound healing, while oxHA produced by H2O2 does not. Taken together, our results show that HA fragmentation by ROS can alter the physiological activity of HA by altering its MW and structure.
... Glycocalyx thinning occurs due to a decrease in the content of its main components-hyaluronan and heparan sulfate [71], whose biosynthesis rate by endotheliocytes decreases at low shear stress [69]. A decrease in glycocalyx thickness due to its fragmentation has been noted under ROS hyperproduction during oxidative stress in the process of ischemia and ischemia/reperfusion [72][73][74] and a degradation of glycosaminoglycans by ROS derived from stimulated polymorphonuclear leukocytes has been detected [75]. Glycocalyx destruction was also observed when the level of oxidized LDL in plasma increased [76,77]. ...
... An alternative pathway of oxidative stress development in diabetes associated with the formation of superoxide anion radical and other ROS during the reaction of amino-containing compounds with methylglyoxal (Maillard reaction) [43]; 3. Non-enzymatic formation of methylglyoxal when glucose derivatives are attacked by alkoxyl lipid radicals [43]; 4. Chemical modification of LDL, involving various natural dicarbonyls: MDA, glyoxal, and methylglyoxal [11,24,27,38]; 5. Foam cell formation and preaterosclerotic (lipoidosis) damage of the vascular wall under the action of dicarbonyl-modified LDL [11]; 6. Expression of LOX-1 on endotheliocyte membranes under the action of dicarbonyl-modified LDL [54]; 7. Expression of NADPH-oxidase in endotheliocytes during the accumulation of dicarbonyl-modified LDL and under generation of superoxide anion radical by this enzyme system [54]; 8. Stimulation of endotheliocyte apoptosis by superoxide anion radical and other ROS generated by NADPH-oxidase [50][51][52][53]; 9. Glycocalyx degradation under the action of superoxide anion radical and other ROS [71][72][73][74][75]; 10. Free-radical mechanism of endotheliocyte membrane damage and development of endothelial dysfunction [49][50][51][52][53]. ...
The review presents evidence that the main damage to the vascular wall occurs not from the action of “oxidized” LDL, which contain hydroperoxy acyls in the phospholipids located in their outer layer, but from the action of LDL particles whose apoprotein B-100 is chemically modified with low molecular weight dicarbonyls, such as malondialdehyde, glyoxal, and methylglyoxal. It has been argued that dicarbonyl-modified LDL, which have the highest cholesterol content, are particularly “atherogenic”. High levels of dicarbonyl-modified LDL have been found to be characteristic of some mutations of apoprotein B-100. Based on the reviewed data, we hypothesized a common molecular mechanism underlying vascular wall damage in atherosclerosis and diabetes mellitus. The important role of oxidatively modified LDL in endothelial dysfunction is discussed in detail. In particular, the role of the interaction of the endothelial receptor LOX-1 with oxidatively modified LDL, which leads to the expression of NADPH oxidase, which in turn generates superoxide anion radical, is discussed. Such hyperproduction of ROS can cause destruction of the glycocalyx, a protective layer of endotheliocytes, and stimulation of apoptosis in these cells. On the whole, the accumulated evidence suggests that carbonyl modification of apoprotein B-100 of LDL is a key factor responsible for vascular wall damage leading to atherogenesis and endothelial dysfunction. Possible ways of pharmacological correction of free radical processes in atherogenesis and diabetogenesis are also discussed.
... These data suggest that dermal inflammation in CKD5 patients is quantitatively associated with loss of hyaluronan (Hijmans et al., 2019). In the present study we found an association between plasma lymphocytes and tissue UEA1 suggesting an association between glycocalyx breakdown and lymphocytopenia, thus with inflammation which is in accordance with published research (Moseley et al., 1997;Vigetti et al., 2010;Oberleithner et al., 2011;Vlahu et al., 2012). In the (above-mentioned) study of Vlahu et al. (2012), the eGC breakdown was indeed related to inflammation in dialysis patients with elevated CRP values (>10 mg/L) having significantly increased perfused boundary regions. ...
... Subsequently, fluid permeability increased . More in vitro studies reported reactive oxygen species and inflammatory cytokines such as tumor necrosis factor-α to lead to hyaluronan degradation in the glycocalyx of endothelial cells and polymorphonuclear leukocytes (Moseley et al., 1997;Vigetti et al., 2010). Importantly, inflammation might also be linked to (and probably induced by) overhydration as has been shown by this work and other researchers [reviewed by Colombo et al. (2008)]. ...
Cardiovascular morbidity is a major problem in patients with chronic kidney disease (CKD) and endothelial dysfunction (ED) is involved in its development. The luminal side of the vascular endothelium is covered by a protective endothelial glycocalyx (eGC) and indirect evidence indicates eGC loss in CKD patients. We aimed to investigate potential eGC loss and ED in skin biopsies of CKD patients and their association with inflammation and volume overload. During living kidney transplantation procedure, abdominal skin biopsies were taken from 11 patients with chronic kidney disease stage 5 of whom 4 were treated with hemodialysis and 7 did not receive dialysis treatment. Nine healthy kidney donors served as controls. Biopsies were stained and quantified for the eGC marker Ulex europaeus agglutinin-1 (UEA1) and the endothelial markers vascular endothelial growth factor-2 (VEGFR2) and von Willebrand factor (vWF) after double staining and normalization for the pan-endothelial marker cluster of differentiation 31. We also studied associations between quantified log-transformed dermal endothelial markers and plasma markers of inflammation and hydration status. Compared to healthy subjects, there was severe loss of the eGC marker UEA1 ( P < 0.01) while VEGFR2 was increased in CKD patients, especially in those on dialysis ( P = 0.01). For vWF, results were comparable between CKD patients and controls. Skin water content was identical in the three groups, which excluded dermal edema as an underlying cause in patients with CKD. The dermal eGC/ED markers UEA1, VEGFR2, and vWF all associated with plasma levels of NT-proBNP and sodium (all R ² > 0.29 and P < 0.01), except for vWF that only associated with plasma NT-proBNP. This study is the first to show direct histopathological evidence of dermal glycocalyx loss and ED in patients with CKD. In line with previous research, our results show that ED associates with markers of volume overload arguing for strict volume control in CKD patients.
... The formation of ROS and acetaldehyde adducts makes conjugated proteins more susceptible to proteolysis [15]. ROS, separately, are capable of degrading some of the glycoconjugates [69]. ROS and hydroxyl radicals in particular have been demonstrated to be capable of the degradation of the major extracellular matrix components: collagen, proteoglycans and glycosaminoglycans (Figure 3). ...
... Non-sulphated glycosaminoglycans (GAGs) such as hyaluronian and chondroitin are the most susceptible to ROS fragmentation. Moseley et al. [69] even proposed that lysosomal glycosidases, derived from polymorphonuclear leukocytes, exert no degradational effect upon intact degradation of glycoconjugates by ROS. This effect is thought to occur via the random modification to the unit monosaccharides, followed by the hydrolytic cleavage of the unstable constituents. ...
The relationship between alcohol consumption and glycoconjugate metabolism is complex and multidimensional. This review summarizes the advances in basic and clinical research on the molecular and cellular events involved in the metabolic effects of alcohol on glycoconjugates (glycoproteins, glycolipids, and proteoglycans). We summarize the action of ethanol, acetaldehyde, reactive oxygen species (ROS), nonoxidative metabolite of alcohol — fatty acid ethyl esters (FAEEs), and the ethanol-water competition mechanism, on glycoconjugate biosynthesis, modification, transport and secretion, as well as on elimination and catabolism processes. As the majority of changes in the cellular metabolism of glycoconjugates are generally ascribed to alterations in synthesis, transport, glycosylation and secretion, the degradation and elimination processes, of which the former occurs also in extracellular matrix, seem to be underappreciated. The pathomechanisms are additionally complicated by the fact that the effect of alcohol intoxication on the glycoconjugate metabolism depends not only on the duration of ethanol exposure, but also demonstrates dose- and regional-sensitivity. Further research is needed to bridge the gap in transdisciplinary research and enhance our understanding of alcohol- and glycoconjugate-related diseases.
... In health, the production of ROS and RNS is constantly reduced by the antioxidant system, but in a pathological state such as cardiac surgery, the imbalance occurs in favor of oxidation, which promotes proteolysis and glycocalyx shedding, and increases vascular permeability and endothelial dysfunction [62]. Endothelial degradation mediated by ROS and RNS can occur directly by the activation of enzymes that shed components of the endothelial glycocalyx, predominantly chondroitin, heparan sulfate, dermatan sulfate, and hyaluronic acid [63,64],or indirectly via activation of MMPs and inhibition of the endogenous antioxidant system [47,65]. In addition to endothelial degradation caused by inflammation, glycocalyx shedding during cardiac surgery can be a result of ischemia-reperfusion injury [66]. ...
Cardiac surgery is one of the highest-risk procedures, usually involving cardiopulmonary bypass and commonly inducing endothelial injury that contributes to the development of perioperative and postoperative organ dysfunction. Substantial scientific efforts are being made to unravel the complex interaction of biomolecules involved in endothelial dysfunction to find new therapeutic targets and biomarkers and to develop therapeutic strategies to protect and restore the endothelium. This review highlights the current state-of-the-art knowledge on the structure and function of the endothelial glycocalyx and mechanisms of endothelial glycocalyx shedding in cardiac surgery. Particular emphasis is placed on potential strategies to protect and restore the endothelial glycocalyx in cardiac surgery. In addition, we have summarized and elaborated the latest evidence on conventional and potential biomarkers of endothelial dysfunction to provide a comprehensive synthesis of crucial mechanisms of endothelial dysfunction in patients undergoing cardiac surgery, and to highlight their clinical implications.
... For instance, an attack on hyaluronan of joint synovial fluid by the hydroxyl radicals in activated neutrophils causes a degradation of this glycosaminoglycan (GAG) (Greenwald & Moak, 1986;Grootveld et al., 1991;McCord, 1974). The hyaluronan strand that is composed of a linear repeating disaccharide →4)-β-D-GlcpA-(1→3)-β-D-GlcpNAc-(1→ units is cleaved through β-scission of the radicals formed either at C(1) of the monosaccharide ring, at C(3) of the N-acetylglucosamine, or at C(4) of the glucuronic acid (Moseley, Waddington, & Embery, 1997;Rees & Davies, 2006;Rees, Hawkins, & Davies, 2004). This ROS-mediated hyaluronan degradation is linked to a reduced viscosity of synovial fluid in the rheumatoid arthritis. ...
... MMPs can promote the degradation of collagen fibrous networks and weaken the stability and durability of dentin-adhesive bonding (Montagner et al., 2014;Perdigão et al., 2013;Tjäderhane et al., 2013). On the other hand, histochemical studies have discovered that bleaching agents also damage proteoglycans in the organic dentin matrix and interfered with the maintenance of interfibrillar spaces, potentially hindering diffusion between adhesives and collagen networks (Bartold et al., 1984;Briso et al., 2014;Moseley et al., 1997). Therefore, the conventional application of antioxidants after bleaching treatment may not be ideal for reversing the decreased dentin bonding, especially in terms of aged bond strength. ...
This in vitro study aimed to investigate the effect of quercetin pretreatment on the bond strength of bleached dentin. Human dentin blocks (2 × 2 × 1 mm) were prepared and randomly divided into 5 groups (n = 16): deionized water pretreatment + no bleaching treatment (DNB); deionized water pretreatment + bleaching treatment (DYB); 75 μg/mL quercetin pretreatment + bleaching (Q75B); 150 μg/mL quercetin pretreatment + bleaching (Q150B); and 300 μg/mL quercetin pretreatment + bleaching (Q300B). The surfaces of superficial dentin (bonding surfaces) were treated with the respective solutions for 2 min, and then the surfaces opposite to the bonding surfaces (near pulp, bleaching surfaces) were subjected to bleaching treatment with 40% hydrogen peroxide (Ultradent, USA) for two 15-min sessions (groups DYB, Q75B, Q150B, and Q300B). After the bleaching procedure, the bonding surfaces were bonded with resin cements (Panavia V5, Kuraray, Japan). The bonded specimens were then divided into 2 subgroups (n = 8): the aging group (subgroup T), which was subjected to 10,000 thermocycles, and the nonaging group (subgroup N), which was not subjected to thermocycling. The microshear bond strength (μSBS) was obtained using a universal testing machine (AGS-X, Shimadzu, Tokyo, Japan). Additional dentin blocks (5 × 5 × 1 mm) were prepared and treated the same as the groups DYB, Q75B, Q150B, and Q300B (n = 8) to evaluate the color change, defined as groups CCDYB, CCQ75B, CCQ150B, and CCQ300B, respectively. Color evaluation was performed using a spectrophotometer (Vita Easyshade Advance 4.0, Vident, USA) to obtain a baseline and again at the end of the bleaching treatment. The data were analyzed via two-way analysis of variance (ANOVA) and Tukey's post-hoc test (α = 0.05). For the immediate bond strength, the specimens in the groups Q75B, Q150B, and Q300B showed significantly higher μSBS values than those in the group DYB (all P < 0.05). No significant differences in the μSBS values were found among the groups Q75B, Q150B, Q300B, and DNB, respectively (all P > 0.05). For the aged bond strength, both the groups Q150B and Q300B exhibited significantly higher μSBS values than groups DYB and DNB (all P < 0.05), whereas no significance differences were found between groups Q150B and Q300B (P = 1.00) or between the groups DYB and DNB (P = 1.00). No significant differences were observed in the △E values among all the groups tested (P = 0.80). Therefore, the application of quercetin for 2 min prior to the bleaching procedure preserved the immediate bond strength and improved the aged bond strength of bleached dentin while maintaining the effectiveness of bleaching.
... In vitro exposure of ROS (superoxide and hydroxyl radicals) to the eGCX promotes fragmentation of GAGs and loss of some of its components. Previous studies have demonstrated that hyaluronan and chondroitin sulfate are the most susceptible to depolymerization and chemical modifications by ROS (Halliwell, 1978;Greenwald and Moy, 1980;Bartold et al., 1984;Moseley et al., 1995Moseley et al., , 1997Lipowsky and Lescanic, 2013;Singh et al., 2013). Intact eGCX has the capability to quench free radicals by having binding sites for anti-oxidant enzymes like xanthine oxidoreductase (Adachi et al., 1993) and endothelial superoxide dismutase (eSOD) (Becker et al., 1994). ...
Expressed on the endothelial cell (EC) surface of blood vessels, the glycocalyx (GCX), a mixture of carbohydrates attached to proteins, regulates the access of cells and molecules in the blood to the endothelium. Besides protecting endothelial barrier integrity, the dynamic microstructure of the GCX confers remarkable functions including mechanotransduction and control of vascular tone. Recently, a novel perspective has emerged supporting the pleiotropic roles of the endothelial GCX (eGCX) in cardiovascular health and disease. Because eGCX degradation occurs in certain pathological states, the circulating levels of eGCX degradation products have been recognized to have diagnostic or prognostic values. Beyond their biomarker roles, certain eGCX fragments serve as pathogenic factors in disease progression. Pharmacological interventions that attenuate eGCX degradation or restore its integrity have been sought. This review provides our current understanding of eGCX structure and function across the microvasculature in different organs. We also discuss disease or injury states, such as infection, sepsis and trauma, where eGCX dysfunction contributes to severe inflammatory vasculopathy.
... They have been linked to oxidative stress and inflammatory response in OA [24]. ROS degrades a variety of GAGs, such as chondroitin sulfate (CS), hyaluronic acid (HA), and dermatan sulfate [25]. Potential sources of ROS identified in chondrocytes, such as NO synthase, can modulate chondrocyte behavior and extracellular matrix homeostasis in OA patients. ...
Osteoarthritis (OA) is a chronic degenerative joint disease characterized by the deterioration of articular cartilage. The progression of OA leads to an increase in inflammatory mediators in the joints, thereby promoting the destruction of the cartilage matrix. Recent studies have reported on the anti-inflammatory and antioxidant properties of cardamonin, which also appears to interact with cellular targets, such as nuclear erythroid 2-related factor 2 (Nrf2), extracellular signal-regulated kinase (ERK), and mammalian target of rapamycin (mTOR) during the progression of tumors. To date, few studies have investigated the effects of cardamonin on chondrocyte inflammation. In the current study, we determined that treating interleukin-1 beta (IL-1β-stimulated chondrocyte cells) with cardamonin significantly reduced the release of nitric oxide (NO) and prostaglandin E2 (PGE2) and significantly inhibited the expression of pro-inflammatory proteins, including inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Cardamonin was also shown to: (1) inhibit the activation and production of matrix metalloproteinases (MMPs), (2) suppress the nuclear factor-κB (NF-κB) signaling pathway, (3) suppress the expression of toll-like receptor proteins, (4) activate the Nrf2 signaling pathway, and (5) increase the levels of antioxidant proteins heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). The increase in antioxidant proteins led to corresponding antioxidant effects (which were abolished by Nrf2 siRNA). Our findings identify cardamonin as a candidate Nrf2 activator for the treatment and prevention of OA related to inflammation and oxidative stress.
... Under pathological conditions such as elevated inflammation and oxidative stress, degradation of sialic acid caps occurs leaving neurons and other cells with a damaged glycocalyx (184)(185)(186). During such pathological conditions, soluble sialic acid residues actually accumulate in serum signifying the removal of sialic acid caps from glycoproteins (184,185). ...
The retina is a complex tissue with multiple cell layers that are highly ordered. Its sophisticated structure makes it especially sensitive to external or internal perturbations that exceed the homeostatic range. This necessitates the continuous surveillance of the retina for the detection of noxious stimuli. This task is mainly performed by microglia cells, the resident tissue macrophages which confer neuroprotection against transient pathophysiological insults. However, under sustained pathological stimuli, microglial inflammatory responses become dysregulated, often worsening disease pathology. In this review, we provide an overview of recent studies that depict microglial responses in diverse retinal pathologies that have degeneration and chronic immune reactions as key pathophysiological components. We also discuss innovative immunomodulatory therapy strategies that dampen the detrimental immunological responses to improve disease outcome.
... Окислительный стресс при сепсисе также играет значимую роль в повреждении ЭГ. In vitro было показано, что выброс супероксидных радикалов и гидроксильных радикалов приводит к фрагментации гликозаминогликанов с последующей утратой части их компонентов [39,40]. При деградации ЭГ клетки эндотелия подвергаются окислительному стрессу, который сопровождается увеличением пористости и проницаемости сосудов и интерстициальными потерями альбумина. ...
Glycocalyx is a gel-like layer covering the surface of vascular endothelial cells. It consists of membrane-attached proteoglycans, glycosaminoglycan chains, glycoproteins, and plasma adhesive proteins. Glycocalyx plays a key role in maintaining vascular homeostasis, controls vascular permeability and the tone of the microvasculature, prevents microvascular thrombosis and regulates leukocyte adhesion. In sepsis and septic shock, damage and shedding of glycocalyx occurs. The degradation of glycocalyx is activated by reactive oxygen species and pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 в (IL-1 в). The inflammation-mediated degradation of glycocalyx leads to vascular hyperpermeability, unregulated vasodilation, microvascular thrombosis and enhanced leukocyte adhesion. Clinical studies have demonstrated a correlation between the levels of glycocalyx components in the blood and organ dysfunction and mortality in sepsis and septic shock. Inflammation-induced damage to glycocalyx can cause a number of specific clinical effects of sepsis, including acute kidney damage, respiratory failure and liver dysfunction. Infusion therapy is an integral part of the treatment of sepsis, but super-aggressive infusion load methods (leading to hypervolemia) may increase the degradation of glycocalyx. Moreover, some markers of glycocalyx degradation, such as circulating levels of syndecan 1 or heparan sulfate, can be used as markers of endothelial dysfunction and sepsis severity.
... Systemic inflammation, modeled by LPS, increases permeability of the BBB (Varatharaj & Galea, 2017). There are several potential mechanisms for this finding including endothelial damage and alteration in beta-catenin, ZO-1, and claudin-5 expression (Cardoso et al., 2012); degradation of the glycocalyx mediated by TNF and ROS (Moseley, Waddington, & Embery, 1997;Wiesinger et al., 2013); astrocyte loss and structural changes (Biesmans et al., 2013); and astrocyte gene transcription with a proinflammatory profile (Zamanian et al., 2012). Interestingly, there seems to be a specific window in brain development when the BBB is susceptible to systemic inflammation since systemic inflammation, modeled by intraperitoneal injection of LPS, induced increased BBB permeability was only observed in rats before the postnatal age of 20 days (Stolp, Dziegielewska, Ek, Potter, & Saunders, 2005). ...
Dysbiosis of the gut microbiome in preterm infants predisposes the neonate to various major morbidities including neonatal necrotizing enterocolitis and sepsis in the neonatal intensive care unit, and adverse neurological outcomes later in life. There are parallel early developmental windows for the gut microbiota and the nervous system during prenatal to postnatal of life. Therefore, preterm infants represent a unique population in which optimization of initial colonization and microbiota development can affect brain development and enhance neurological outcomes. In this review, we will first discuss the factors affecting the assembly of neonatal gut microbiota and the contribution of dysbiosis in preterm infants to neuroinflammation and neurodevelopmental disorders. We then will discuss the emerging pathways connecting the gut microbiome and brain development. Further we will discuss the significance of current models for alteration of the gut microbiome (including humanized gnotobiotic models and exposure to antibiotics) to brain development and functions. Understanding the role of early optimization of the microbiome in brain development is of paramount importance for developing microbiome‐targeted therapies and protecting infants from prematurity‐related neurodevelopmental diseases.
... Thrombin, cathepsin B, elastase, proteinase 3, and plasmin have all been found to be capable of releasing hyaluronic acid from the glycocalyx, for example, from isolated, perfused guinea pig hearts (Becker, Jacob et al. 2015); although these enzymes were found to be far less potent than hyaluronidase. It also could be considered that reactive oxygen species (ROS) could be at least partially responsible for a diabetes-induced loss of hyaluronic acid from the glycocalyx, given the role of ROS in diabetic retinopathy (Eshaq, Wright et al. 2014) and their specific targeting of hyaluronic acid compared with sulfated glycosaminoglycans (Moseley, Waddington et al. 1997). ...
We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW = 155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW = 376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.
... 162,163 Both superoxide and peroxynitrite cause chemical fragmentation of HA to generate HA fragments (LMW HA) that act as endogenous danger signals that interact with HA and TLRs to activate the innate immune system. [164][165][166][167][168] Interestingly, we and others have demonstrated that HA fragments are able to activate the NLRP3 inflammasome, 152,153 thereby providing direct rationale to study the effects of HAbinding peptides in models of BPD, and eventually in preterm infants at risk of developing BPD. These concepts are depicted in Figure 2. ...
Over 50 years after its first description, Bronchopulmonary Dysplasia (BPD) remains a devastating pulmonary complication in preterm infants with respiratory failure and develops in 30–50% of infants less than 1000-gram birth weight. It is thought to involve ventilator- and oxygen-induced damage to an immature lung that results in an inflammatory response and ends in aberrant lung development with dysregulated angiogenesis and alveolarization. Significant morbidity and mortality are associated with this most common chronic lung disease of childhood. Thus, any therapies that decrease the incidence or severity of this condition would have significant impact on morbidity, mortality, human costs, and healthcare expenditure. It is clear that an inflammatory response and the elaboration of growth factors and cytokines are associated with the development of BPD. Numerous approaches to control the inflammatory process leading to the development of BPD have been attempted. This review will examine the anti-inflammatory approaches that are established or hold promise for the prevention or treatment of BPD.
... Le glycocalyx a été retrouvé altéré dans de nombreuses situations associées à un stress oxydant accrue tel que le diabète 113 , l'hémodialyse 115 , l'ischémie-reperfusion 54 . Un effet direct des espèces réactives de l'oxygène (ROS) sur la dégradation des GAG a pu être mis en évidence dans une étude effectuée sur des cellules en culture 105 . De même l'effet néfaste de ces espèces radicalaires est suggéré par le rôle protecteur d'agents captant les ROS comme la superoxyde dysmutase (SOD) 74 3. ...
Le glycocalyx est un déterminant essentiel de la physiologie endothéliale vasculaire. En effet, il limite l'activation de la coagulation et il adapte la perfusion capillaire. Son altération dans les pathologies auto-immunes vasculaires où une dysfonction endothéliale est présente comme le syndrome des antiphospholipides (SAPL) et la sclérodermie systémique (ScS) semble probable. L'apparition récente du microscan pourrait offrir une meilleure évaluation du glycocalyx notamment dans le SAPL. Enfin, le microscan permet de coupler l'évaluation du glycocalyx à celle de la microcirculation ce qui nous a conduit à réaliser également une étude préliminaire chez des patients atteints de ScS. MATÉRIEL ET MÉTHODES. Au cours de la première étude portant sur les patients atteints d'un SAPL artériel primaire, une évaluation biochimique du glycocalyx par dosage du syndecan-1 et de l'acide hyaluronique sérique chez 15 patients SAPL et chez 17 contrôles appariés a été réalisée. Nous disposions pour ces deux groupes d'une estimation de l'épaisseur intima-média, d'une évaluation de la dilation médiée par le flux (FMD), d'un dosage de marqueur du stress oxydant (les TBARS) et du dosage de l'activité du facteur tissulaire. L'épaisseur du glycocalyx a été mesurée au niveau des capillaires sublinguaux à l'aide d'un microscanie. Dans la seconde étude portant sur les patients sclérodermiques, outre la mesure du glycocalyx, une évaluation microcirculatoire comportant une vidéo capillaroscopie et une évaluation sublinguale par microscan® a été réalisée chez 16 patients ScS et 10 contrôles. RÉSULTATS. Les taux de syndecan-1 étaient plus élevés chez les patients atteints de SAPL (39.3±19.5 ig/m1 vs 19.1±13 tig/m1; p<0.01) avec une corrélation négative entre ces taux et la FMD (1.2=0.47 ; p<0.001) et une corrélation positive avec l'épaisseur intima média (r2=0.2 ; p=0.01) et le facteur tissulaire (r40.25 ; p<0.01). L'épaisseur du glycocalyx était diminuée chez les patients atteints de SAPL (0.14±0.16p.m vs 0.79+0.26p.m ; p<0.01). L'épaisseur du glycocalyx était également diminuée chez les patients suivis pour ScS par rapport aux témoins (0.40±0.21gm vs 0.76±0.29pin ; p=0.003). Au niveau microcirculatoire, une altération de la densité capillaire sublinguale par rapport aux témoins était retrouvée (10.56+1.35 vs 11.58+0.85 ; p= 0.04). La densité capillaire était corrélée avec la densité capillaire péri-unguéale (r2= 0.36 ; p=0.03). CONCLUSION. Cette étude retrouve pour la première fois une altération du glycocalyx au cours du SAPL. Elle retrouve une altération couplée du glycocalyx et de la microcirculation sublinguale pour la ScS. Ces évaluations endothéliales et microcirculatoires non-invasives par le microscan pourraient s'étendre à d'autres pathologies auto-immunes. Enfin, elles pourraient permettre d'objectiver leur évolution sous traitement.
Obesity imposes deficits to adipose tissue and vascular endothelium, yet the role that distinct adipose depots play in mediating endothelial dysfunction in local arteries remains unresolved. We recently showed that obesity impairs endothelial Kir2.1 channels, mediators of NO production, in arteries of visceral adipose tissue (VAT) while Kir2.1 function in subcutaneous adipose tissue (SAT) endothelium remains intact. Therefore, we determined if VAT vs. SAT from lean or diet-induced obese mice affected Kir2.1 channel function in vitro. We found that VAT from obese mice reduces Kir2.1 function without altering channel expression whereas AT from lean mice and SAT from obese mice had no effect on Kir2.1 function as compared to untreated control cells. As Kir2.1 is well-known to be inhibited by fatty acid derivatives and obesity is strongly associated with elevated circulating fatty acids, we next tested the role of the fatty acid translocase CD36 in mediating VAT-induced Kir2.1 dysfunction. We found that downregulation of CD36 restored Kir2.1 currents in endothelial cells exposed to VAT from obese mice. In addition, endothelial cells exposed to VAT from obese mice exhibited a significant increase in CD36-mediated fatty acid uptake. The importance of CD36 in obesity-induced endothelial dysfunction of VAT arteries was further supported in ex vivo pressure myography studies where CD36 ablation rescued the endothelium-dependent response to flow via restoring Kir2.1 and eNOS function. These findings provide new insight into the role of VAT in mediating obesity-induced endothelial dysfunction and suggest a novel role for CD36 as a mediator of endothelial Kir2.1 impairment.
Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cannot be discarded based on the results of the present study.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40635-023-00548-w.
Neutrophilic polymorphonuclear leukocytes (neutrophils) are myeloid cells packed with lysosomal granules (hence also called granulocytes) that contain a formidable antimicrobial arsenal. They are terminally differentiated cells that play a critical role in acute and chronic inflammation, as well as in the resolution of inflammation and wound healing. Neutrophils express a dense array of surface receptors for multiple ligands, ranging from integrins to support their egress from bone marrow into the circulation and from the circulation into tissues, to cytokine/chemokine receptors that drive their navigation to the site of infection or tissue damage and also prime them for a second stimulus, to pattern recognition receptors and immunoglobulin receptors to facilitate the destruction and removal of infective agents or debridement of damaged tissues. When afferent neutrophil signals are proportionate and coordinated they will phagocytose opsonized and unopsonized bacteria, activating the nicotinamide adenine dinucleotide phosphate oxidase (respiratory burst) to generate reactive oxygen species, which augment the proteolytic destruction of microbes secured within the phagosome. A highly orchestrated process of apoptosis follows, forming membrane‐bound substructures that are removed by macrophages. Neutrophils are capable of various other forms of programmed cell death, such as NETosis and pyroptotic cell death, as well as nonprogrammed cell death by necrosis. In recent years, research has revealed that neutrophils are capable of far more subtle cell‐cell interactions than previously thought possible. This includes synthesis of various inflammatory mediators and also myeloid cell training within bone marrow, where epigenetic and metabolic signals associated with returning neutrophils that undergo reverse egress from tissues into the vasculature and back to bone marrow program a hyperreactive subset of neutrophils during myelopoiesis that are capable of hypersensitive reactions to microbial aggressors. These characteristics are evident in various neutrophil subsets/subpopulations, creating broad heterogeneity in the behavior and biological repertoire of these seemingly schizophrenic immune cells. Moreover, neutrophils are critical effector cells of adaptive and innate immunity, binding to opsonized bacteria and destroying them by extracellular and intracellular methods. The former creates substantial collateral host tissue damage, as they are less specific than T‐cytotoxic cell‐killing mechanisms, and in conditions such as peri‐implantitis, where plasma cells and neutrophils dominate the immune infiltrate, bone and tissue destruction are rapid and appear relentless. Finally, the role of neutrophils as conduits for periodontal‐systemic disease connections and for oxidative damage to act as a causal link between the two has only recently been realized. In this chapter, we attempt to expand on these issues, emphasizing the contributions of European scientists throughout a detailed appraisal of the benefits and side effects of neutrophilic inflammation and immune function.
The endothelial glycocalyx (EG) is a gel-like structure that forms a layer in between the surface of the endothelium and lumen. EG was once thought to be merely a structural support for the endothelium. However, in recent years, the importance of EG as a first line of defense and a key regulator to endothelial integrity has been illuminated. With advanced age, EG deterioration becomes more noticeable and at least partially associated with endothelial dysfunction. Hyaluronan (HA), one of the critical components of the EG, has distinct properties and roles to the maintenance of EG and endothelial function. Therefore, given the intimate relationship between the EG and endothelium during the aging process, HA may serve as a promising therapeutic target to prevent endothelial dysfunction.
While fluid resuscitation is fundamental in the treatment of sepsis-induced tissue hypo-perfusion, a sustained positive fluid balance is associated with excess mortality. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water, has not been tested previously as adjuvant to fluid resuscitation in sepsis.
In a prospective, parallel-grouped, blinded model of porcine peritonitis-sepsis, we randomized animals to intervention with adjuvant hyaluronan (add-on to standard therapy) (n = 8) or 0.9% saline (n = 8). After the onset of hemodynamic instability the animals received an initial bolus of 0.1 % hyaluronan (1 mg/kg/10 min) or placebo (0.9% saline) followed by a continuous infusion of 0.1% hyaluronan (1 mg/kg/h) or saline during the experiment. We hypothesized that the administration of hyaluronan would reduce the volume of fluid administered (aiming at stroke volume variation <13%) and/or attenuate the inflammatory reaction.
Total volumes of intravenous fluids infused were 17.5 ± 11 ml/kg/h vs. 19.0 ± 7 ml/kg/h in intervention and control groups, respectively ( p = 0.442). Plasma IL-6 increased to 2450 (1420 – 6890) pg/ml and 3690 (1410 – 11960) pg/ml (18 hours of resuscitation) in the intervention and control groups (NS). The intervention counteracted the increase in proportion of fragmented hyaluronan associated with peritonitis-sepsis (mean peak elution fraction (18 hours of resuscitation) intervention group: 16.8 ± 0.9 vs. control group: 17.9 ± 0.6 ( p = 0.031)).
In conclusion, hyaluronan did not reduce the volume needed for fluid resuscitation or decrease the inflammatory reaction, even though it counterbalanced the peritonitis induced shift towards increased proportion of fragmented hyaluronan.
It remains uncertain how brain glycosaminoglycans (GAGs) contribute to the progression of inflammatory disorders like multiple sclerosis (MS). We investigated here neuroinflammation-mediated changes in GAG composition and metabolism using the mouse model of experimental autoimmune encephalomyelitis (EAE) and sham-immunized mice as controls. Cerebellum, mid- and forebrain at different EAE phases were investigated using gene expression analysis (microarray and RT-qPCR) as well as HPLC quantification of CS and hyaluronic acid (HA). The cerebellum was the most affected brain region showing a downregulation of Bcan, Cspg5, and an upregulation of Dse, Gusb, Hexb, Dcn and Has2 at peak EAE. Upregulation of genes involved in GAG degradation as well as synthesis of HA and decorin persisted from onset to peak, and diminished at remission, suggesting a severity-related decrease in CS and increments in HA. Relative disaccharide quantification confirmed a 3.6 % reduction of CS-4S at peak and a normalization during remission, while HA increased in both phases by 26.1 % and 17.6 %, respectively. Early inflammatory processes led to altered GAG metabolism in early EAE stages and subsequent partially reversible changes in CS-4S and in HA. Targeting early modifications in CS could potentially mitigate progression of EAE/MS.
Periodontitis is one of the most prevalent infectious inflammatory diseases, characterized by irreversible destruction of the supporting tissues of teeth, which is correlated with a greater risk of multiple systemic diseases, thus regarded as a major health concern. Dysregulation between periodontal microbial community and host immunity is considered to be the leading cause of periodontitis. Comprehensive studies have unveiled the double-edged role of immune response in the development of periodontitis. Immune senescence, which is described as age-related alterations in immune system, including a diminished immune response to endogenous and exogenous stimuli, a decline in the efficiency of immune protection, and even failure in immunity build-up after vaccination, leads to the increased susceptibility to infection. Recently, the intimate relationship between immune senescence and periodontitis has come into focus, especially in the aging population. In this review, both periodontal immunity and immune senescence will be fully introduced, especially their roles in the pathology and progression of periodontitis. Furthermore, novel immunotherapies targeting immune senescence are presented to provide potential targets for research and clinical intervention in the future.
The glycocalyx is a polysaccharide structure that protrudes from the body of a cell. It is primarily conformed of glycoproteins and proteoglycans, which provide communication, electrostatic charge, ionic buffering, permeability, and mechanosensation-mechanotransduction capabilities to cells. In blood vessels, the endothelial glycocalyx that projects into the vascular lumen separates the vascular wall from the circulating blood. Such a physical location allows a number of its components, including sialic acid, glypican-1, heparan sulfate, and hyaluronan, to participate in the mechanosensation-mechanotransduction of blood flow-dependent shear stress, which results in the synthesis of nitric oxide and flow-mediated vasodilation. The endothelial glycocalyx also participates in the regulation of vascular permeability and the modulation of inflammatory responses, including the processes of leukocyte rolling and extravasation. Its structural architecture and negative charge work to prevent macromolecules greater than approximately 70 kDa and cationic molecules from binding and flowing out of the vasculature. This also prevents the extravasation of pathogens such as bacteria and virus, as well as that of tumor cells. Due to its constant exposure to shear and circulating enzymes such as neuraminidase, heparanase, hyaluronidase, and matrix metalloproteinases, the endothelial glycocalyx is in a continuous process of degradation and renovation. A balance favoring degradation is associated with a variety of pathologies including atherosclerosis, hypertension, vascular aging, metastatic cancer, and diabetic vasculopathies. Consequently, ongoing research efforts are focused on deciphering the mechanisms that promote glycocalyx degradation or limit its syntheses, as well as on therapeutic approaches to improve glycocalyx integrity with the goal of reducing vascular disease. © 2022 American Physiological Society. Compr Physiol 12: 1-31, 2022.
Cancer propagation and progression are associated with remarkable remodeling of the extracellular microenvironment with the formation of a permissive matrix for tumor growth, enriched in inflammatory mediators and matrix-degrading enzymes. Proteoglycans, which represent major structural and functional extracellular matrix components, play pivotal roles by affecting tissue organization, cell–matrix interactions, cell signaling, and, ultimately, cell behavior in physiological conditions as well as during cancer development and progression. In this respect, the proteolytic activity in tumor stroma liberates bioactive fragments, which can be detected in serum and may be useful as diagnostic and prognostic markers. Furthermore, since cell surface proteoglycans are specifically expressed by cancer cells, as in the case of glypican-3 in hepatocellular carcinoma, they can represent potential promising targets for immunotherapy. This chapter reviews and critically addresses the major clinical literature and provides an overview of the significance of circulating proteoglycans (PGs) and glycosaminoglycans (GAGs), especially their soluble forms originating from the cell surface heparan sulfate proteoglycans syndecans and glypicans, in both prognosis and diagnosis of various malignancies responsible for high mortality and disability, including hepatocellular carcinoma, multiple myeloma, breast cancer, and renal cell carcinoma.
The surface of all animal cells is coated with a layer of carbohydrates linked in various ways to the outer side of the plasma membrane. These carbohydrates are mainly bound to proteins in the form of glycoproteins and proteoglycans and together with the glycolipids constitute the so-called glycocalyx. In particular, the endothelial glycocalyx that covers the luminal layer of the endothelium is composed of glycosaminoglycans (heparan sulphate -HS and hyaluronic acid -HA), proteoglycans (syndecans and glypicans) and adsorbed plasma proteins. Thanks to its ability to absorb water, this structure contributes to making the surface of the vessels slippery but at the same time acts by modulating the mechano-transduction of the vessels, the vascular permeability and the adhesion of leukocytes in thus regulating several physiological and pathological events. Among the various enzymes involved in the degradation of the glycocalyx, heparanase (HPSE) has been shown to be particularly involved. This enzyme is responsible for the cutting of heparan sulfate (HS) chains at the level of the proteoglycans of the endothelial glycocalyx whose dysfunction appears to have a role in organ fibrosis, sepsis and viral infection.
In this mini-review, we describe the mechanisms by which HPSE contributes to glycocalyx remodeling and then examine the role of glycocalyx degradation in the development of pathological conditions and pharmacological strategies to preserve glycocalyx during disease pathogenesis.
The glycocalyx is a ubiquitous structure found on endothelial cells that extends into the vascular lumen. It is enriched in proteoglycans, which are proteins attached to the glycosaminoglycans heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. In health and disease, the endothelial glycocalyx is a central regulator of vascular permeability, inflammation, coagulation, and circulatory tonicity. During sepsis, a life-threatening syndrome seen commonly in hospitalized patients, the endothelial glycocalyx is degraded, significantly contributing to its many clinical manifestations. In this review we discuss the intrinsically linked mechanisms responsible for septic endothelial glycocalyx destruction: glycosaminoglycan degradation and proteoglycan cleavage. We then examine the consequences of local endothelial glycocalyx loss to several organ systems and the systemic consequences of shed glycocalyx constituents. Last, we explore clinically relevant non-modifiable and modifiable factors that exacerbate or protect against endothelial glycocalyx shedding during sepsis.
Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.
Polysaccharides can be elite carriers for therapeutic molecules due to their versatility and low probability to trigger toxicity and immunogenic responses. Local and systemic therapies can be achieved through particle pulmonary delivery, a promising non-invasive alternative. Successful pulmonary delivery requires particles with appropriate flowability to reach alveoli and avoid premature clearance mechanisms. Polysaccharides can form micro-, nano-in-micro-, and large porous particles, aerogels, and hydrogels. Herein, the characteristics of polysaccharides used in drug formulations for pulmonary delivery are reviewed, providing insights into structure-function relationships. Charged polysaccharides can confer mucoadhesion, whereas the ability for specific sugar recognition may confer targeting capacity for alveolar macrophages. The method of particle preparation must be chosen considering the properties of the components and the delivery device to be utilized. The fate of polysaccharide-based carriers is dependent on enzyme-triggered hydrolytic and oxidative mechanisms, allowing their complete degradation and elimination through urine or reutilization of released monosaccharides.
The 8-hydroxy-deoxyguanosine (8-OHdG) is one of the customary products of oxidized DNA. The purpose of this study was to compare salivary and plasma 8-OHdG concentrations in a group of chronic periodontitis patients to those measured in a group of patients with healthy periodontium, as well as to determine the impact of periodontal therapy on 8-OHdG concentrations in saliva and plasma in chronic periodontitis patients. The study sample comprised of 24 patients with chronic periodontitis and 16 periodontally healthy individuals. Plaque index, gingival index, papilla bleeding index, probing depth and clinical attachment level were indices used to determine patient periodontal status. Salivary and plasma 8-OHdG concentrations were determined by ELISA method. The salivary 8-OHdG concentration was statistically significantly higher in the group of periodontitis patients compared to periodontally healthy subjects. After initial periodontal therapy, the 8-OHdG concentration in saliva was significantly reduced in the periodontitis group (p=0.021). Differences in plasma 8-OHdG concentrations between the two groups did not reach statistical significance and no significant changes were noted in the periodontitis group following initial periodontal therapy. A higher salivary 8-OHdG concentration reflects increased oxidative stress caused by periodontal disease. Initial periodontal therapy may be helpful in reducing salivary 8-OHdG concentrations in chronic periodontitis patients.
Glioblastoma (GBM) is uniformly fatal with a median survival of 1–2 years, despite best available treatments including, radiotherapy (RT), and chemotherapy (CHT). Despite continued advances in image-guided hypofractionated stereotactic radiosurgery, GBM remains one of the most invasive tumors with 100% recurrence rate. Multiple biological processes contribute to the development of resistance to cytotoxic chemotherapy, and perpetuation of cell molecular changes which cause treatment failure, tumor cell escape, and reestablishment of new tumor foci in the brain. Impacts of prior brain RT in GBM pathophysiology are less understood, though increasing evidence suggests RT-induced changes in the brain microenvironment contribute to recurrent GBM phenotype. The tumor microenvironment impacts malignant cells directly and indirectly through stromal cells and stroma-GBM cell interactions that support tumor progression. Changes in extracellular matrix (ECM), abnormal vasculature, hypoxia, and inflammation have been reported to promote tumor aggressiveness that could be exacerbated by prior RT. Prior radiation may have long-term impacts on microglia and brain-infiltrating monocytes, leading to lasting alterations in cell and cytokine signaling which can cause irreversible changes in ECM, thus affecting the extracellular distribution of small molecules, and chemotherapeutic drugs making them sparsely available to combat tumor growth. Since RT is vital to GBM care, but substantially alters the tumor microenvironment, we here discuss the challenges, knowledge gaps, and therapeutic opportunities relevant to targeting protumorigenic features of the GBM microenvironment. Understanding the intricate biological processes involved can allow us to incorporate new targeted interventions in current treatment approaches and during postprimary treatment regimens early enough to overcome the setting of secondary tumor growth. We suggest that insights gained from RT-induced changes in the tumor microenvironment may provide opportunities for development of new combinatorial therapies that could help better manage the late effects of RT, which otherwise get amplified in previously radiated brain microenvironment making its stroma permissive for recurrent tumors that are chemo-radiotherapy resistant and cause poor outcome in glioblastoma.
Cell-surface heparan sulfate proteoglycans (HSPGs) play key roles in regulating cell behavior, cell signaling, and cell matrix interactions in both physiological and pathological conditions. Their soluble forms from glycocalyx shedding are not merely waste products, but, rather, bioactive molecules, detectable in serum, which may be useful as diagnostic and prognostic markers. In addition, as in the case of glypican-3 in hepatocellular carcinoma, they may be specifically expressed by pathological tissue, representing promising targets for immunotherapy. The primary goal of this comprehensive review is to critically survey the main findings of the clinical data from the last 20 years and provide readers with an overall picture of the diagnostic and prognostic value of circulating HSPGs. Moreover, issues related to the involvement of HSPGs in various pathologies, including cardiovascular disease, thrombosis, diabetes and obesity, kidney disease, cancer, trauma, sepsis, but also multiple sclerosis, preeclampsia, pathologies requiring surgery, pulmonary disease, and others will be discussed.
The endothelial glycocalyx, the gel layer covering the endothelium, is composed of glycosaminoglycans, proteoglycans, and adsorbed plasma proteins. This structure modulates vessels’ mechanotransduction, vascular permeability, and leukocyte adhesion. Thus, it regulates several physiological and pathological events. In the present review, we described the mechanisms that disturb glycocalyx stability such as reactive oxygen species, matrix metalloproteinases, and heparanase. We then focused our attention on the role of glycocalyx degradation in the induction of profibrotic events and on the possible pharmacological strategies to preserve this delicate structure.
Introduction:
Hemorrhagic shock has recently been shown to cause shedding of a carbohydrate surface layer of endothelial cells known as the glycocalyx. This shedding of the glycocalyx is thought to be a mediator of the coagulopathy seen in trauma patients. Clinical studies have demonstrated increases in shed glycocalyx in the blood after trauma, and animal studies have measured glycocalyx disruption in blood vessels in the lung, skeletal muscle, and mesentery. However, no study has measured glycocalyx disruption across a wide range of vascular beds to quantify the primary locations of this shedding.
Methods:
In the present study, we used a rat model of hemorrhagic shock and resuscitation to more comprehensively assess glycocalyx disruption across a range of organs. Glycocalyx disruption was assessed by fluorescent-labelled wheat germ agglutinin or syndecan-1 antibody staining in flash frozen tissue.
Results:
We found that our model did elicit glycocalyx shedding, as assessed by an increase in plasma syndecan-1 levels. In tissue sections, we found that the greatest glycocalyx disruption occurred in vessels in the lung and intestine. Shedding to a lesser extent was observed in vessels of the brain, heart, and skeletal muscle. Liver vessel glycocalyx was unaffected, and kidney vessels, including the glomerular capillaries, displayed an increase in glycocalyx. We also measured reactive oxygen species (ROS) in the endothelial cells from these organs, and found that the greatest increase in ROS occurred in the two beds with the greatest glycocalyx shedding, the lungs and intestine. We also detected fibrin deposition in lung vessels following hemorrhage-resuscitation.
Conclusions:
We conclude that the endothelium in the lungs and intestine are particularly susceptible to the oxidative stress of hemorrhage-resuscitation, as well as the resulting glycocalyx disruption. Thus these two vessel beds may be important drivers of coagulopathy in trauma patients.
Chondroitin sulfate (CS), the main component of cartilage extracellular matrix, has attracted attention as a biomaterial for cartilage tissue engineering. However, current CS hydrogel systems still have limitations for application in successful cartilage tissue engineering owing to their unsuitable degradation kinetics, insufficient mechanical similarity, and lack of integration with the native cartilage tissue. In this study, using mussel adhesive-inspired catechol chemistry, we developed a functional CS hydrogel that exhibits tunable physical and mechanical properties as well as excellent tissue adhesion for efficient integration with native tissues. Various properties of the developed catechol-functionalized CS (CS-CA) hydrogel, including swelling, degradation, mechanical properties, and adhesiveness, could be tailored by varying the conjugation ratio of the catechol group to the CS backbone and the concentration of the CS-CA conjugates. CS-CA hydrogels exhibited significantly increased modulus (∼10 kPa) and superior adhesive properties (∼3 N) over conventional CS hydrogels (∼hundreds Pa and ∼0.05 N). In addition, CS-CA hydrogels incorporating decellularized cartilage tissue dice promoted the chondrogenic differentiation of human adipose-derived mesenchymal stem cells by providing a cartilage-like microenvironment. Finally, the transplantation of autologous cartilage dice using tissue-adhesive CS-CA hydrogels enhanced cartilage integration with host tissue and neo-cartilage formation owing to favorable physical, mechanical, and biological properties for cartilage formation. In conclusion, our study demonstrated the potential utility of the CS-CA hydrogel system in cartilage tissue reconstruction.
Peridontal hastalıklar dişin destek dokularında enflasyonla ve yıkımla karakterize enfeksiyöz hastalıklardır. Periodontal hastalığın ilerlemesinde, bakteriyel kolonizasyona karşı gelişen immünolojik reaksiyonlar önemli rol oynamaktadır. Periodontal hastalıklar ile kardiovasküler hastalıkların temel etkeni olan ateroskleroz arasındaki direk nedensel ilişki tam olarak belirlenememesine karşın, her iki hastalığı ilişkilendiren farklı patolojik mekanizmalar ve ortak risk faktörleri mevcuttur. Aterosklerotik plakların periodontal patojenlerle enfeksiyonu, periodontal enflamasyonun kronik sistemik enflamasyon yoluyla aterojenik etki oluşturması her iki hastalığın olası ilişkisini açıklayan olası mekanizmalardandır. Serbest radikaller ile koruyucu antioksidan sistem arasındaki dengenin bozulması sonucu meydana gelen oksidatif stresin, son yıllarda her iki hastalığın başlaması ve ilerlemesiyle ilişkili enflamatuar bir belirteç olduğu üzerinde durulmaktadır. Bu derlemede periodontal hastalık, ateroskleroz ve oksidatif stres arasındaki ilişkinin ele alınması amaçlanmıştır.
The intra‐articular injection of high molecular weight hyaluronic acid (HA) has been reported to be an effective treatment for pain of osteoarthritis of the knee. However, the mechanism by which HA exerts its effect is unknown. To explore HA's influence on the growth of U937 human macrophages, cells were incubated for 168 h with three concentrations, 1, 0.1 and 0.01 mg/mL, of Hyalgan®, a high molecular weight HA preparation. At 24‐h increments, the cells were examined for proliferation, cell cycle distribution as well as the number of apoptotic and dead cells. Exposing macrophages to 1 mg/mL Hyalgan® significantly reduced the rate of cellular proliferation and altered the cell cycle distribution to yield decreased proportions of G0/G1 cells but increased S and G2/M cells. Concomitantly, a 10‐fold increase in apoptotic cells and a 12‐fold increase in dead cells were observed. The population doubling time (PDT) for cells treated with 1.0 mg/mL Hyalgan® increased from 23.6 to 52.9 h. By contrast, the two lower Hyalgan® concentrations significantly promoted macrophage proliferation in a dose‐dependent manner. They also increased the proportion of G2/M cells, but had no effect on the number of apoptotic or dead cells. The PDTs of 21.5 and 22.2 h were less than the control time of 23.6 h. These results demonstrate that Hyalgan® concentrations have a differential effect on macrophage growth dynamics and suggest an anti‐inflammatory effect at high HA concentrations. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality.
Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in articular cartilage and the loss of CS-GAG occurs early in OA. As a major component of perichondral matrix interacting directly with chondrocytes, the active turnover of CS can affect to break the homeostasis of chondrocytes. Here we employ CS-based 3-dimensional (3D) hydrogel scaffold system to investigate how the degradation products of CS affect the catabolic phenotype of chondrocytes. The breakdown of CS-based ECM by the chondroitinase ABC (ChABC) resulted in a hypertrophy-like morphologic change in chondrocytes, which was accompanied by catabolic phenotypes, including increased MMP-13 and ADAMTS5 expression, nitric oxide (NO) production and oxidative stress. The inhibition of Toll-like receptor 2 (TLR2) or TLR4 with OxPAPC (TLR2 and TLR4 dual inhibitor) and LPS-RS (TLR4-MD2 inhibitor) ameliorated these catabolic phenotypes of chondrocytes by CS-ECM degradation, suggesting a role of CS breakdown products as damage-associated molecular patterns (DAMPs). As downstream signals of TLRs, MAP kinases, NF-kB, NO and STAT3-related signals were responsible for the catabolic phenotypes of chondrocytes associated with ECM degradation. NO in turn reinforced the activation of MAP kinases as well as NFkB signaling pathway. Thus, these results propose that the breakdown product of CS-GAG can recapitulate the catabolic phenotypes of OA.
Vascular endothelial cells line the inner surface of the entire cardiovascular system as a single layer and are involved in an impressive array of functions, ranging from the regulation of vascular tone in resistance arteries and arterioles, modulation of microvascular barrier function in capillaries and postcapillary venules, and control of proinflammatory and prothrombotic processes, which occur in all segments of the vascular tree but can be especially prominent in postcapillary venules. When tissues are subjected to ischemia/reperfusion (I/R), the endothelium of resistance arteries and arterioles, capillaries, and postcapillary venules become dysfunctional, resulting in impaired endothelium-dependent vasodilator and enhanced endothelium-dependent vasoconstrictor responses along with increased vulnerability to thrombus formation, enhanced fluid filtration and protein extravasation, and increased blood-to-interstitium trafficking of leukocytes in these functionally distinct segments of the microcirculation. The number of capillaries open to flow upon reperfusion also declines as a result of I/R, which impairs nutritive perfusion. All of these pathologic microvascular events involve the formation of reactive species (RS) derived from molecular oxygen and/or nitric oxide. In addition to these effects, I/R-induced RS activate NLRP3 inflammasomes, alter connexin/pannexin signaling, provoke mitochondrial fission, and cause release of microvesicles in endothelial cells, resulting in deranged function in arterioles, capillaries, and venules. It is now apparent that this microvascular dysfunction is an important determinant of the severity of injury sustained by parenchymal cells in ischemic tissues, as well as being predictive of clinical outcome after reperfusion therapy. On the other hand, RS production at signaling levels promotes ischemic angiogenesis, mediates flow-induced dilation in patients with coronary artery disease, and instigates the activation of cell survival programs by conditioning stimuli that render tissues resistant to the deleterious effects of prolonged I/R. These topics will be reviewed in this article.
Cell-matrix interactions are fundamental to many developmental, homeostatic, immune and pathologic processes. Hyaluronan (HA), a critical component of the extracellular matrix (ECM) that regulates normal structural integrity and development, also regulates tissue responses during injury, repair, and regeneration. Though simple in its primary structure, HA regulates biological responses in a highly complex manner with balanced contributions from its molecular size and concentration, synthesis versus enzymatic and/or oxidative-nitrative fragmentation, interactions with key HA binding proteins and cell associated receptors, and its cell context-specific signaling. This review highlights the different, but inter-related factors that dictate the biological activity of HA and introduces the overarching themes that weave throughout this special issue of Matrix Biology on hyaluronan.
Background:
The blood-brain barrier acts as a highly regulated interface; its dysfunction may exacerbate, and perhaps initiate, neurological and neuropsychiatric disorders.
Methods:
In this narrative review, focussing on redox, inflammatory and mitochondrial pathways and their effects on the blood-brain barrier, a model is proposed detailing mechanisms which might explain how increases in blood-brain barrier permeability occur and can be maintained with increasing inflammatory and oxidative and nitrosative stress being the initial drivers.
Results:
Peripheral inflammation, which is causatively implicated in the pathogenesis of major psychiatric disorders, is associated with elevated peripheral pro-inflammatory cytokines, which in turn cause increased blood-brain barrier permeability. Reactive oxygen species, such as superoxide radicals and hydrogen peroxide, and reactive nitrogen species, such as nitric oxide and peroxynitrite, play essential roles in normal brain capillary endothelial cell functioning; however, chronically elevated oxidative and nitrosative stress can lead to mitochondrial dysfunction and damage to the blood-brain barrier. Activated microglia, redox control of which is mediated by nitric oxide synthases and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, secrete neurotoxic molecules such as reactive oxygen species, nitric oxide, prostaglandin, cyclooxygenase-2, quinolinic acid, several chemokines (including monocyte chemoattractant protein-1 [MCP-1], C-X-C motif chemokine ligand 1 [CXCL-1] and macrophage inflammatory protein 1α [MIP-1α]) and the pro-inflammatory cytokines interleukin-6, tumour necrosis factor-α and interleukin-1β, which can exert a detrimental effect on blood-brain barrier integrity and function. Similarly, reactive astrocytes produce neurotoxic molecules such as prostaglandin E2 and pro-inflammatory cytokines, which can cause a 'leaky brain'.
Conclusion:
Chronic inflammatory and oxidative and nitrosative stress is associated with the development of a 'leaky gut'. The following evidence-based approaches, which address the leaky gut and blood-brain barrier dysfunction, are suggested as potential therapeutic interventions for neurological and neuropsychiatric disorders: melatonin, statins, probiotics containing Bifidobacteria and Lactobacilli, N-acetylcysteine, and prebiotics containing fructo-oligosaccharides and galacto-oligosaccharides.
Background:
Superficial erosion currently causes at least one-third of acute coronary syndromes (ACS), and its incidence is increasing. Yet, the underlying mechanisms in humans are still largely unknown.
Objectives:
The authors sought to assess the role of hyaluronan (HA) metabolism in ACS.
Methods:
Peripheral blood mononuclear cells were collected from ACS (n = 66), stable angina (SA) (n = 55), and control (CTRL) patients (n = 45). The authors evaluated: 1) gene expression of hyaluronidase 2 (HYAL2) (enzyme degrading high-molecular-weight HA to its proinflammatory 20-kDa isoform) and of CD44v1, CD44v4, and CD44v6 splicing variants of HA receptor; and 2) HYAL2 and CD44 protein expression. Moreover, they compared HYAL2 and CD44 gene expression in ACS patients with plaque erosion (intact fibrous cap and thrombus) and in ACS patients with plaque rupture, identified by optical coherence tomography analysis.
Results:
Gene expression of HYAL2, CD44v1, and CD44v6 were significantly higher in ACS as compared with SA (p = 0.003, p < 0.001, and p = 0.033, respectively) and CTRL subjects (p < 0.001, p < 0.001, and p = 0.009, respectively). HYAL2 protein expression was significantly higher in ACS than in SA (p = 0.017) and CTRL (p = 0.032), whereas no differences were found in CD44 protein expression. HYAL2 and CD44v6 gene expression was significantly higher in patients with plaque erosion than in those with plaque rupture (p = 0.015 and p = 0.029, respectively).
Conclusions:
HYAL2 and CD44v6 splicing variants seem to play an important role in ACS, in particular when associated with plaque erosion. After further validation, HYAL2 might represent a potentially useful biomarker for the noninvasive identification of this mechanism of coronary instability.
Hyaluronan (HA) is a large polymer and an important component of the extracellular matrix. During homeostasis, high molecular mass HA is the predominant form, but upon inflammation, degradation products of HA accumulate. These HA fragments (HA-fs) have been reported to possess pro-inflammatory activities and thus act as alarmins, notifying immune cells of danger via TLR4 and CD44. HA is found in large quantities in synovial joint fluid. In order to reveal a potential role of HA-fs in arthritis pathogenesis, the in vitro effects of HA of various molecular masses (from 1680 kDa to oligosaccharide HA) on synovial fibroblasts and chondrocytes from rheumatoid arthritis patients, and on peripheral blood mononuclear cells from healthy donors, were investigated. TLR4 and CD44 surface expression was confirmed by immunocytochemistry, and cell activation was determined based on cytokine and chemokine production. While the cell types investigated expressed TLR4 and CD44, no increased release of IL-1ß, IL-6, IL-8, IL-10, IL-12 or TNF-α was detected after HA stimulation. Similarly, HA did not enhance activation after priming cells with low doses of LPS or by forming complexes with LPS. Hence, this study does not support the common view of HA-fs being pro-inflammatory mediators and it is not likely that HA-fs generated during arthritis contribute to disease pathogenesis.
The glycosaminoglycan hyaluronan (HA) is a key component of the microenvironment surrounding cells. In healthy tissues, HA molecules have extremely high molecular mass and consequently large hydrodynamic volumes. Tethered to the cell surface by clustered receptor proteins, HA molecules crowd each other, as well as other macromolecular species. This leads to severe nonideality in physical properties of the biomatrix, because steric exclusion leads to an increase in effective concentration of the macromolecules. The excluded volume depends on both polymer concentration and hydrodynamic volume/molecular mass. The biomechanical properties of the extracellular matrix, tissue hydration, receptor clustering, and receptor-ligand interactions are strongly affected by the presence of HA and by its molecular mass. In inflammation, reactive oxygen and nitrogen species fragment the HA chains. Depending on the rate of chain degradation relative to the rates of new synthesis and removal of damaged chains, short fragments of the HA molecules can be present at significant levels. Not only are the physical properties of the extracellular matrix affected, but the HA fragments decluster their primary receptors and act as endogenous danger signals. Bioanalytical methods to isolate and quantify HA fragments have been developed to determine profiles of HA content and size in healthy and diseased biological fluids and tissues. These methods have potential use in medical diagnostic tests. Therapeutic agents that modulate signaling by HA fragments show promise in wound healing and tissue repair without fibrosis.
Background: Periodontal disease is common chronic adult condition. Antioxidants are present in the body fluid as protection against free radical. Uric acid is one of antioxidants that can be found in saliva. Moreover, the relationship among the antioxidant enzymes activities and clinical periodontal status were investigated. Objectives: The aim of the study was to observe uric acid level activities in the saliva of gingivitis and periodontitis patients. Methods: Six patients with gingivitis and six patients with periodontitis in Dental Hospital Trisakti University were included in the study. Clinical condition of each subject, the plaque index, and probing depth were determined. The salivary uric acid level was measured using the Folin-Wu method. Result: Salivary uric acid levels in the periodontitis patients with a mean ± SD 7.40 ± 0.31 (p = 0.004) were found to be higher compared to the gingivitis patients (mean ± SD = 6.84 ± 0.19). In addition, there were no significant differences in salivary uric acid levels between gender (p = 0.641). Conclusion: Uric acid levels in periodontitis patients were found to be higher than in gingivitis patients. Moreover, uric acid has more role on periodontitis than in gingivitis as an antioxidant agent.
In the vascular system, the endothelial surface layer (ESL) as the inner surface of blood vessels affects mechanotransduction, vascular permeability, rheology, thrombogenesis, and leukocyte adhesion. It creates barriers between endothelial cells and blood and neighbouring cells. The glycocalyx, composed of glycoconjugates and proteoglycans, is an integral component of the ESL and a key element in inter- and intracellular communication and tissue homeostasis. In pathophysiological conditions (atherosclerosis, infection, ischemia/reperfusion injury, diabetes, trauma and acute lung injury) glycocalyx-degrading factors, i.e. reactive oxygen and nitrogen species, matrix metalloproteinases, heparanase and sialidases, damage the ESL, thereby impairing endothelial functions. This leads to increased capillary permeability, leucocyte-endothelium interactions, thrombosis and vascular inflammation, the latter further driving glycocalyx destruction. The present review highlights current knowledge on the vasculoprotective role of the ESL, with specific emphasis on its remodelling in inflammatory vascular diseases and discusses its potential as a novel therapeutic target to treat vascular pathologies.
Objectives:
The aim of this study was to estimate tissue and gingival crevicular fluid (GCF) levels of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) in premenopausal, perimenopausal and postmenopausal women with chronic periodontitis.
Background:
Oxidative stress has been implicated in the etiopathogenesis of periodontitis and menopause induces oxidative stress.
Materials and methods:
According to Stages of Reproductive Aging Workshop (STRAW) criteria, women diagnosed with periodontitis were subdivided into three groups of 31 participants each 1. Premenopausal 2. Perimenopausal and 3. Postmenopausal. GCF and gingival tissue samples were collected from sites with maximum probing depth. Tissue DNA was extracted from the gingival sample and 8-OHdG in the extracted DNA, and GCF samples were measured using ELISA.
Results:
There was a highly significant difference in the overall GCF 8-OHdG levels among the three groups with the pairwise difference being highly significant between the premenopausal-postmenopausal groups and perimenopausal-postmenopausal groups. However, no overall significant differences in tissue 8-OHdG levels were found among the three groups. Pairwise, highly significant differences were found between the premenopausal-postmenopausal groups and perimenopausal-postmenopausal groups for tissue 8-OHdG levels. No significant correlations were found between various measure of periodontal disease and GCF/tissue 8-OHdG levels among all the groups.
Conclusion:
Premenopausal-postmenopausal and perimenopausal-postmenopausal transition resulted in significant increase in tissue and GCF 8-OHdG levels. However, no association was found between stages of reproductive ageing and tissue levels of 8-OHdG.
Objective
Expression of Spam1/PH20 and its modulation of high/low molecular weight hyaluronan substrate have been proposed to play an important role in murine oligodendrocyte precursor cell (OPC) maturation in vitro and in normal and demyelinated central nervous system (CNS). We reexamined this using highly purified PH20.
Methods
Steady‐state expression of mRNA in OPCs was evaluated by quantitative polymerase chain reaction; the role of PH20 in bovine testicular hyaluronidase (BTH) inhibition of OPC differentiation was explored by comparing BTH to a purified recombinant human PH20 (rHuPH20). Contaminants in commercial BTH were identified and their impact on OPC differentiation characterized. Spam1/PH20 expression in normal and demyelinated mouse CNS tissue was investigated using deep RNA sequencing and immunohistological methods with two antibodies directed against recombinant murine PH20.
Results
BTH, but not rHuPH20, inhibited OPC differentiation in vitro. Basic fibroblast growth factor (bFGF) was identified as a significant contaminant in BTH, and bFGF immunodepletion reversed the inhibitory effects of BTH on OPC differentiation. Spam1 mRNA was undetected in OPCs in vitro and in vivo; PH20 immunolabeling was undetected in normal and demyelinated CNS.
Interpretation
We were unable to detect Spam1/PH20 expression in OPCs or in normal or demyelinated CNS using the most sensitive methods currently available. Further, “BTH” effects on OPC differentiation are not due to PH20, but may be attributable to contaminating bFGF. Our data suggest that caution be exercised when using some commercially available hyaluronidases, and reports of Spam1/PH20 morphogenic activity in the CNS may be due to contaminants in reagents.
Diabetes induces the onset and progression of renal injury through causing hemodynamic dysregulation along with abnormal morphological and functional nephron changes. The most important event that precedes renal injury is an increase in permeability of plasma proteins such as albumin through a damaged glomerular filtration barrier resulting in excessive urinary albumin excretion (UAE). Moreover, once enhanced UAE begins, it may advance renal injury from progression of abnormal renal hemodynamics, increased glomerular basement membrane (GBM) thickness, mesangial expansion, extracellular matrix accumulation, and glomerulosclerosis to eventual end-stage renal damage. Interestingly, all these pathological changes are predominantly driven by diabetes-induced reactive oxygen species (ROS) and abnormal downstream signaling molecules. In diabetic kidney, NADPH oxidase (enzymatic) and mitochondrial electron transport chain (nonenzymatic) are the prominent sources of ROS, which are believed to cause the onset of albuminuria followed by progression to renal damage through podocyte depletion. Chronic hyperglycemia and consequent ROS production can trigger abnormal signaling pathways involving diverse signaling mediators such as transcription factors, inflammatory cytokines, chemokines, and vasoactive substances. Persistently, increased expression and activation of these signaling molecules contribute to the irreversible functional and structural changes in the kidney resulting in critically decreased glomerular filtration rate leading to eventual renal failure.
During the past few years, a considerable number of studies have examined different aspects of the host response in gingival crevicular fluid (GCF), including the relationship of specific markers to the active phases of periodontal disease. Various indicators of the acute inflammatory response (the lysosomal enzymes P-glucuronidase and collagenase, the cytoplasmic enzyme aspartate aminotransferase, and the arachidonic acid metabolite PGE2) have been shown to be associated with clinical attachment loss in chronic adult periodontitis in man and experimental periodontitis in animal models. In contrast, the relationship of indicators of the humoral immune response in GCF to active periodontal disease is equivocal. Furthermore, a number of indicators of the cellular immune response have been identified recently in GCF (i.e., Interleukin-la, IL-1β, tumor necrosis factor-a), but their relationship to active phases of periodontal disease have not been studied.
The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.
The oxidative reductive depolymerization (ORD) of hyaluronate has been investigated. A solution of hyaluronate (Mr 4.07 x 10(5] in phosphate buffer (pH 7.2) was incubated in the presence of Fe2+ for 24 h at 37 degrees C under an oxygen atmosphere to yield depolymerized hyaluronate (ORD fragments; an average Mr of 2,600). The ORD fragments contain 21 and 24% less hexosamine and uronic acid, respectively, but no olefinic linkage. They were exhaustively digested with chondroitinase AC-II. The resulting oligosaccharides and monosaccharides were separated by gel filtration and ion-exchange chromatography, and their structures were determined by proton and carbon-13 NMR, fast atom bombardment mass spectrometry, and chromatographic techniques combined with chemical modifications. The following structures derived from the reducing ends of the ORD fragments were identified: 4,5-unsaturated GlcA(beta 1----3)-N-acetyl-D-glucosaminic acid (where GlcA- represents glucuronosyl-) (21%), 4,5-unsaturated GlcA(beta 1----3)GlcNAc(beta 1----3)-D-arabo-pentauronic acid (24%), and N-acetyl-D-glucosamine (51%). The following structures derived from the nonreducing ends were identified: L-threo-tetro-dialdosyl-(1----3)GlcNAc (a tentative structure, 8%), N-acetylhyalobiuronic acid (20%), and N-acetyl-D-glucosamine (45%). The results indicate that the ORD reaction of hyaluronate proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents.
The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.
The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2∸ and H2O2 supported these generalfindings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient by persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2∸ production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2∸ and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2∸ for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2∸ and H2O2. The failure of the CL assay to report O2∸ and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2∸ fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2∸ and H2O2 generated by either stimulated neutrophils or in cell-free systems.
Superoxide anion, a highly reactive free radical, was generated in vitro using enriched purified xanthine oxidase. Collagen solutions exposed to superoxide radical failed to gel normally when heated to 37 degrees C. The magnitude of the inhibition of gelation was propotional to duration of exposure and to flux of superoxide. Since inhibition of collagen gelation reflects alteration of collagen biochemistry, and/or collagen degradation, it is suggested that the action of free radicals produced in vivo by leukocytes may adversely affect the structural or functional integrity of cartilage and adjacent joint structures.
A solution of hog intestinal heparin (average M(r) 12,000, anti-clotting activity 168 USP units/mg) in 0.2 M phosphate buffer (pH 7.2), was incubated in the presence of Fe2+ for 20 h at 50 degrees under an O2 atmosphere to yield oxidative-reductively depolymerized heparin (ORD heparin, average M(r) 3,000, anti-clotting activity 34 USP units/mg). Chemical analysis of the ORD heparin showed a 22, 26, and 14% loss of hexosamine, uronic acid, and N-acetyl group, respectively, but no remarkable loss of both total and N-sulfate groups. 1H and 13C NMR spectroscopic analysis indicated no decrease in the amount of L-iduronic acid 2-sulfate, but a marked loss of nonsulfated uronic acid (73 and 39% loss of D-glucuronic acid and L-iduronic acids, respectively, the sum of which corresponds to the chemically determined loss of total uronic acid). The results indicated that the ORD reaction of heparin proceeds essentially by destruction of monosaccharide units, except L-iduronic acid 2-sulfate residues, due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable residues.
Several statistical models that have been suggested in the periodontal literature for describing longitudinal attachment level changes, such as the gradual loss, single-burst, multiple-burst, and random walk models as well as other models introduced in this paper are compared by their power to predict future attachment loss. The data used in this analysis is from 1061 sites of 8 subjects, with moderate to severe periodontal disease, monitored monthly for about a year. This study found that none of the suggested models could significantly outperform the naïve mean predictor, which predicts the future attachment level from the past mean. It was also found that no single model, such as the burst, gradual, or random walk, together with measurement error can fully explain the variation in the data. These results indicate that in the course of one year, the attachment level change may not follow the same model. Consequently, a model that fits well to past data cannot be accurately extended to the future.
Interaction of bone and dentin proteins with minerals is an elementary step in the regulation of mineralization in these tissues. Adsorption of acidic non-collagenous proteins on hydroxyapatite was examined using fluorescence-labeled protein and synthetic hydroxyapatite. Phosphophoryn, bone Gla protein, osteonectin and bone small proteoglycan II were prepared and labeled with fluorescein. All of these proteins were adsorbed on hydroxyapatite with a dissociation constant on the order of 10(-7) M. The more acidic proteins had lesser binding capacities. Hydroxyapatite single crystals were incubated with labeled proteins and observed with a fluorescence microscope. Phosphophoryn and other acidic proteins were adsorbed preferentially on the (100) face of the crystal. This preferential adsorption of the acidic proteins may be responsible for the morphogenesis of biological hydroxyapatite.
In the gingival crevicular fluid (GCF) of control and chronic adult periodontitis (CAP) patients there is a spontaneous release of O2- radicals from polymorphonuclear leukocytes (PMN). The addition of the exogenous stimuli phorbol myristate acetate (PMA) decreased the O2-. formation in control GCF, while in CAP patients produced a marked enhancement of O2-. generation. The circulating PMN of control subjects did not show a spontaneous O2-. formation, differently from CAP patients. On the contrary, a similar O2-. production was measured when the circulating PMN were stimulated with PMA. Moreover, the antioxidant activity measured in 10 microliters of cell free gingival supernatant (GS) of control and CAP patients had the same values by inhibiting 12.6% and 18.9% respectively of the O2- formation supported by a xanthine/xanthine oxidase system. Probably, the protective or destructive effect of PMN in GCF of CAP patients depends on the variations of the rate of O2- formation in respect to the intrinsic antioxidant property of GS.
During the period covered by this issue, over 500 papers were published in the area of biology of dental hard tissues. This review is limited to publications that focus on the organic matrix components believed to be important in the formation of dentin, cementum, and enamel. The advances made in this area for the collagen-based dental hard tissues (dentin and cementum) have been primarily in the isolation and partial characterization of the noncollagenous proteins such as proteoglycans, phosphoproteins, and proteins normally found in bone. These proteins are being studied because of their potential role for directing hydroxyapatite nucleation or crystal growth. The progress made in the enamel field has been primarily in the area of molecular biology. Enamel is quite different physically from dentin and cementum because it is formed from a noncollagenous matrix (mostly amelogenin), which is almost completely removed and replaced with hydroxyapatite. Serum proteins have now been found in all dental hard tissues including enamel. Opinions on the clinical significance of these advances are provided.
Proteoglycans were extracted from EDTA-demineralized human alveolar bone under dissociative conditions using 4 M guanidinium chloride in the presence of protease inhibitors. The extract was further purified by anion-exchange chromatography on DEAE-Sephacel, using a step-wise salt gradient. The proteoglycan-rich fraction was analysed for carbohydrate, protein and amino acid composition and molecular size by SDS-PAGE. Glycosaminoglycan content was determined by cellulose acetate electrophoresis after proteolysis. The sulphate isomers of the glycosaminoglycans were confirmed by Fourier-transformed infra-red spectroscopy. Two chondroitin sulphate-proteoglycan species were identified with molecular weights of 79 and 55-65 kDa, respectively. The core proteins had molecular weights of 49 kDa for both proteoglycans, with the amino acid content rich in glycine, leucine, glutamate and aspartate. The chondroitin sulphate chains were mainly as the 4-sulphate isomer forms although low but detectable amounts of 6-sulphate isomer were also present.
The control of potentially periodontopathic microorganisms by host neutrophils is crucial to periodontal health. Neutrophils may use oxidative or nonoxidative mechanisms and either kill bacteria, influence bacterial growth, or modify bacterial colonization in the periodontium. Delivery of antimicrobial substances by neutrophils involves respiratory burst activity, phagocytosis, secretion, or cytolysis/apoptosis. Neutrophils contain a number of antimicrobial components including calprotectin complex, lysozyme, defensins, cofactor-binding proteins, neutral serine proteases, bactericidal/permeability increasing protein, myeloperoxidase, and a NADPH oxidase system. Many of these components are multifunctional and exhibit several mechanisms of antimicrobial activity. When comparisons are made among periodontal bacteria, differences in sensitivity to different components are observed. A hypothesis of specific defense is presented: That specific periodontal diseases can result from the failure of specific aspects of the host immune system (the neutrophil, in particular) in its interaction with specific periodontal pathogens. Failure may be due to phenotypic variation (pleomorphism) within the host or bacterial evasive strategies.
Human polymorphonuclear leukocytes (PMN) were able to generate and release superoxide anions upon stimulation of Streptococcus mutans, Bacteroides gingivalis, and Capnocytophaga ochracea when incubated aerobically but not when incubated anaerobically. Lysozyme release and phagocytosis by PMN were independent of oxygen, and no difference between PMN incubated aerobically or anaerobically was observed (PMN stimulated by B. gingivalis released 7.6% total lysozyme when aerobic and 6.9% when anaerobic). There were variations in lysozyme release and phagocytosis for the three organisms, particularly for phagocytosis. B. gingivalis and C. ochracea yielded lower phagocytosis values than those for S. mutans, e.g., at 1 h 67% of the initial inoculum of S. mutans was phagocytosed (versus only 40% for B. gingivalis). Transmission electron microscopy showed that both S. mutans and B. gingivalis were internalized into classical phagolysosomes. In contrast, C. ochracea showed two forms of internalization; C. ochracea either formed a classical phagolysosome or was tightly bound in the cytoplasm with no surrounding cell membrane. Intracellular killing of S. mutans and C. ochracea was unaffected by anaerobiosis, but killing of C. ochracea was much lower than that of S. mutans (1 x 10(7) to 2 x 10(7) bacteria killed compared with 5.1 x 10(7) bacteria killed at 6 h). In contrast, a greater number of B. gingivalis was killed in the presence of oxygen (5.3 x 10(7) bacteria were killed when aerobically incubated and 1.9 x 10(7) bacteria were killed when anaerobically incubated). These results suggest that the ability to survive anaerobically may enable some bacteria to evade PMN killing; however, abnormal phagocytosis may represent a more efficient way to evade both oxygen-dependent and -independent killing mechanisms, leading to enhanced virulence of the organism.
Thirty patients with adult periodontitis were monitored using an automated periodontal probe for 6 months in order to determine the prevalence of active sites and the overall pattern of disease progression in active sites. The automated probe is capable of measuring probing attachment levels relative to the cemento-enamel junction with better than 0.2 mm of accuracy. The prevalence of disease activity was dependent on the threshold for probing attachment loss used. When the smallest threshold (0.4 mm) was used the prevalence of active disease was 29%, whereas a large threshold (2.4 mm) detected only 2% active sites. Regression analysis of the active sites revealed that 76% of the sites lost probing attachment consistent with a continuous model for disease progression. A small subset of sites demonstrated either bursts of activity or exacerbations and remissions of disease activity.
Oxygen-derived free radicals (ODFR) depolymerized synovial-fluid (SF) hyaluronic acid (HA) when hypoxanthine/xanthine oxidase (HX/XAO) was used as the radical generator. The molecular-weight distribution of ODFR-induced SF HA degradation products was determined using high performance liquid chromatography (HPLC) with TSK 5000 PW or TSK 6000 PW size-exclusion columns and simultaneously using 125I-labelled hyaluronate-binding protein (125I-HABP) assay. The exposure of SF HA to hydroxyl-radical flux resulted in the formation of a degradation product having a molecular weight of 13.5 X 10(3) daltons, from which no further degradation was achieved. If the iron chelator desferrioxamine and hydroxyl-radical scavenger mannitol were present in the reaction mixture, the HA peak decreased by 30-50%, as a result of reaction with superoxide radical and hydrogen peroxide. These results show that superoxide radical and hydroxyl radical may have different modes of action on SF HA. The molecular-weight distribution of serum HA from patients with rheumatoid arthritis varied in different individuals and ranged between 275 X 10(3) and 650 X 10(3).
The purpose of this investigation was to study the proteoglycans in alveolar bone of three animal species. Alveolar bone was obtained from humans, pigs, and rabbits. Portions were fixed, sectioned, and stained with monoclonal antibodies against keratan sulfate and chondroitin sulfate. In other samples, biochemical analyses were performed. After removal of the organic matrix by 4 mol/L guanidinium HCl extraction in the presence of proteinase inhibitors, proteoglycans in the mineralized matrix were extracted with 4 mol/L guanidinium HCl/0.5 mol/L EDTA/proteinase inhibitors, and characterized on the basis of their glycosaminoglycan content (cellulose acetate membrane electrophoresis), charge (DEAE-Sephacel and hydroxylapatite chromatography), size (Sepharose CL-6B chromatography and agarose/polyacrylamide gel electrophoresis), and amino acid content. The results indicated that keratan sulfate could be detected immunohistochemically and biochemically in rabbit bone only. The predominant glycosaminoglycan in pig and human alveolar bone was chondroitin sulfate, although some hyaluronate, dermatan sulfate, and heparan sulfate were also detected. The proteoglycans were found to be slightly smaller than gingival proteoglycans, but similar to those in cementum, dentin, and other bones. In addition to intact proteoglycans, some free glycosaminoglycan chains were also extracted from the mineralized matrix. Amino acid analyses showed some subtle differences between alveolar bone proteoglycan and those of the soft tissues of the periodontium.
Decorin is a small chondroitin-dermatan sulphate proteoglycan consisting of a core protein and a single glycosaminoglycan chain. Eighty per cent of the core protein consists of 10 repeats of a leucin-rich sequence of 24 amino acids. Similar repeats have been found in two other proteoglycans, biglycan and fibromodulin, and in several other proteins including Drosophila morphogenetic proteins. Expression of high levels of decorin in Chinese hamster ovary cells has a dramatic effect on their morphology and growth properties. We now report that this effect is due at least in part to the ability of decorin to bind transforming growth factor-beta, an autocrine factor that stimulates the growth of Chinese hamster ovary cells. As transforming growth factor-beta induces synthesis of decorin in many cell types, our results suggest that decorin may be a component of a feedback system regulating cell growth.
A disparity has been noted between the flux of superoxide generated by neutrophils and the effects of those radicals on a typical macromolecular substrate, hyaluronic acid (HA). To further investigate this phenomenon, comparative degradation studies were conducted using phorbol myristate acetate (PMA), which caused neutrophils to readily degrade HA, and digitonin, a stimulus which, like PMA, produced a sustained superoxide flux but which did not affect HA. The differential effects of the 2 stimuli could be related to concomitant release of peroxidase activity. The implications of this mechanism for in vivo oxygen radical effects are discussed.
Degradation (depolymerization) of hyaluronic acid is readily accomplished by superoxide-ion-generating systems, especially those which beget secondary free radicals. It has been presumed, but not confirmed, that this is the mechanism by which neutrophils might alter synovial fluid viscosity. We have demonstrated, in a neutrophil (PMN) superoxide system, physical disruption of the hyaluronate macromolecule using column chromatography and by measurement of intrinsic viscosity. In addition, comparison of calibrated free radical fluxes between a cell-free superoxide system and a neutrophil system revealed very close parallels in iron requirement, inhibition by free radical scavengers, and magnitude of effect. It is concluded that oxygen-derived free radicals are probably the major, if not sole, mechanism by which neutrophils might degrade hyaluronate.
Partially-reduced forms of dioxygen or "oxy-radicals" (superoxide, O2-/HO2; hydrogen peroxide, H2O2; hydroxyl radical X OH) and oxidants of comparable reactivity are implicated in an increasing number of physiological, toxicological, and pathological states. Transition metal catalysis is recognized as being integral to the generation and the reactions of these activated oxygen species. Factors such as pH and chelation govern the reactivity of the transition metals with dioxygen and "oxy-radicals" and therefore influence the apparent mechanisms by which oxidative damage to phospholipids, DNA, and other biomolecules is initiated. In biological systems the concentrations of redox-active transition metals capable of catalyzing these reactions appears to be relatively low. However, under certain conditions metal storage and transport proteins (ferritin, transferrin, ceruloplasmin, etc.) may furnish additional redox active metals.
Actinobacillus actinomycetemcomitans is a facultative gram-negative microorganism which has been implicated as an etiologic agent in localized juvenile periodontitis and in subacute bacterial endocarditis and abscesses. Although resistant to serum bactericidal action and to oxidant injury mediated by superoxide anion (O2-) and hydrogen peroxide (H2O2), this organism is sensitive to killing by the myeloperoxidase-hydrogen peroxide-chloride system (K.T. Miyasaki, M.E. Wilson, and R.J. Genco, Infect. Immun. 53:161-165, 1986). In this study, we examined the sensitivity of A. actinomycetemcomitans to killing by intact neutrophils under aerobic conditions, under anaerobic conditions, and under aerobic conditions in the presence of the heme-protein inhibitor sodium cyanide. Intact neutrophils killed opsonized A. actinomycetemcomitans under aerobic and anaerobic conditions, and the kinetics of these reactions indicated that both oxidative and nonoxidative mechanisms were operative. Oxidative mechanisms contributed significantly, and most of the killing attributable to oxidative mechanisms was inhibited by sodium cyanide, which suggested that the myeloperoxidase-hydrogen peroxide-chloride system participated in the oxidative process. We conclude that human neutrophils are capable of killing A. actinomycetemcomitans by both oxygen-dependent and oxygen-independent pathways, and that most oxygen-dependent killing requires myeloperoxidase activity.
Patients with localized juvenile periodontitis (LJP) exhibit defective neutrophil functions to a variety of environmental and host stimuli. It is not clear, however, how many of the measurable functions are defective and whether individual patients exhibit single or multiple dysfunctions. The purpose of this study was to evaluate chemotaxis, phagocytosis, specific granule release and superoxide production in a group of 23 previously unreported LJP patients. Our results indicate that all 23 of these LJP patients exhibited chemotaxis depression to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and endotoxin-activated serum (EAS). Smaller groups from the 23 chemotactically defective LJP group were used to test other function due to inability to obtain sufficient quantities of blood. Fourteen of 14 LJP patients tested exhibited defective phagocytosis. Ten LJP patients were evaluated for specific granule release, and 14 LJP patients were evaluated for superoxide production. Both granule release and superoxide production were found to be normal in chemotactically defective LJP patients. Since both defective and normal responses noted in the same neutrophil populations are mediated by the same receptor, it is hypothesized that the cellular defect lies in a post receptor pathway.
Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfate was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate Mr 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated form inflamed tissue was mostly a small molecular weight species which eluted in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.
In 12 patients with juvenile periodontitis (JP), stimulation of peripheral polymorphonuclear neutrophils (PMN) by opsonized bacteria showed increased release of free oxygen radicals and of elastase activity in relation to that of pair-matched healthy controls. The elastase increase was not associated with higher intracellular content of elastase, since the measurement of homogenized PMN cells did not differ between the patient group and the control group; nor did the spontaneous elastase activity of nonstimulated cells differ. Release of elastase by contaminating lymphocytes and platelets could be excluded, since no elastase activity from these cells or interference by these cells could be demonstrated in the assay system used. When formyl-methionyl-leucyl-phenylalanine was used as a stimulant, the chemiluminescence in the patient PMN cells did not differ with statistical significance from their pair-matched controls. The increased release of free oxygen radicals and of elastase seems to be a characteristic of the PMN cells in juvenile periodontitis, indicating hyperactive cells with possible pathogenic effects.
The effect of a chemically induced oxygen-derived free radical flux has been studied on extracted porcine gingival hyaluronic acid and proteoglycans as well as on cryostat sections of porcine gingivae. The result of the free radical producing system on hyaluronic acid and the proteoglycans was one of reduction of specific viscosity and molecular size. When frozen sections were subjected to the same oxygen-derived free radical flux and subsequently stained with Alcian blue, a noticeable decrease in staining intensity was observed when compared to control sections. These results are considered to reflect the in vitro capacity of oxygen-derived free radicals to depolymerize the two major non-fibrous extracellular macromolecules of gingivae. It is postulated that such a mechanism may be responsible, at least in part, for the destruction of gingival proteoglycans and hyaluronic acid observed in inflammatory periodontal disease.
It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.
To investigate possible mechanisms of hyaluronic acid depolymerization, superoxide anion and other secondary oxygen-derived free radicals were generated in vitro and allowed to act upon a hyaluronate substrate. Superoxide, generated either enzymatically with xanthine oxidase or by stimulation of polymorphonuclear leukocytes, reduced the viscosity of hyaluronate solutions dramatically while the chromatographic profiles of the glycosaminoglycan shifted toward lower molecular weights. Superoxide-treated hyaluronate also became susceptible to further degradation by beta-N-acetylglucosaminidase A. Experiments with scavengers of various toxic oxygen-derived free radicals clearly implicated these reactants as mediators of hyaluronate depolymerization. Generation of superoxide by leukocytes in vivo may account for the loss of synovial fluid viscosity that accompanies inflammatory joint disease.
This review presents evidence for lysosomal enzymes being at least partly responsible for the tissue destruction seen in periodontal disease. However, many other inflammatory and immunologic mechanisms have been identified that can contribute to tissue destruction (Nisengard 1977).
The relationship of PMN to the periodontal tissues is equivalent to the proverbial double‐edged sword. The localized tissue destruction that may be due to the extracellular release of PMN enzymes in individuals with normal cell function must be weighed against the role of PMN in containment of gingival bacteria and their products. The consequences of qualitative and/or quantitative abnormalities of neutrophils are far more undesirable for the periodontium. It therefore seems logical to propose that the role of polymorphonuclear leukocytes in the gingival tissues is primarily a defensive one.
The presence of neutrophils in the gingival crevice has been shown to reflect the inflammatory condition of the tissues. Due to their availability it may prove beneficial to use gingival crevicular PMN activity as a reliable clinical index of disease activity. The development of appropriate tests to measure PMN may eventually lead to better diagnostic criteria, including the definition of active versus inactive disease.
The most common forms of destructive periodontal disease have been thought to slowly and continuously progress until treatment or tooth loss. Recently, data have become available which are inconsistent with this "continuous disease" hypothesis. Data from longitudinal monitoring of periodontal attachment levels and alveolar bone in humans and in animals suggest that periodontal disease progresses by recurrent acute episodes. In addition, rates of attachment loss have been measured in individual sites which are faster than those consistent with the continuous disease hypothesis or slower than those expected from estimates of prior loss rates. To account for these observations, a model of destructive periodontal disease is described in which bursts of activity occur for short periods of time in individual sites. These bursts appear to occur randomly at periodontal sites throughout the mouth. Some sites demonstrate a brief active burst of destructive periodontal disease (which could take a few days to a few months) before going into a period of remission. Other sites appear to be free of destructive periodontal disease throughout the individual's life. The sites which demonstrate destructive periodontal activity may show no further activity or could be subject to one or more bursts of activity at later time periods. Comparison of monitored loss rates for a year with mean loss rates prior to monitoring suggested that there may be relatively short periods in an individual's life in which many sites undergo periodontal destruction followed by periods of extended remission. An extension of the random disease model is also suggested in which bursts of destructive periodontal disease activity occur with higher frequency during certain periods of an individual's life.
The effect of reactive oxygen species (ROS) on the chemical structure of glycosaminoglycans (GAG) was studied in order to consider their role in connective tissue damage during an inflammatory disease state such as periodontal disease. GAG were exposed to a radical generating system for 1 h and analysed by gel filtration for fragmentation and chemically with respect to uronic acid, hexosamine and sulfate content. Non-sulfated GAG, hyaluronan and chondroitin, were most susceptible to depolymerisation and chemical modification of uronic acid and hexosamine residues by ROS. Depolymerisation and chemical modification of sulfated GAG, chondroitin 4-sulfate, dermatan sulfate and heparan sulfate was significantly less than for non-sulfated GAG. The highly sulfated GAG heparin showed minimal depolymerisation by ROS, but uronic acid residues were readily modified. Analysis of the ROS-exposed residues suggests that uronic acid is capable of degrading to a 3-carbon aldehyde, malondialdehyde. Chondroitin sulfate exposed to ROS resulted in marginal desulfation. The results suggest that the presence of sulfate on the GAG chain may protect the molecule against ROS attack. However, chemical modification of GAG may affect proteoglycan function and be of importance in considering connective tissue destruction in a variety of pathological situations, including periodontal disease.
The level of malondialdehyde, a stable end product of lipid peroxidation induced by reactive oxygen intermediates and the activity of two potent antioxidant enzymes, superoxide dismutase and glutathione peroxidase, was investigated in tissue homogenates of 22 surgical periapical granuloma specimens. Malondialdehyde levels were significantly higher and glutathione peroxidase activity was significantly lower in periapical granuloma samples than in healthy gingival tissue homogenates, which were used as controls. The activity of superoxide dismutase was similar in periapical granuloma and in control samples. Our results indicate an altered balance between the production and the elimination of toxic oxygen metabolites in chronic apical periodontitis. We hypothesize that reactive oxygen intermediates, which are being produced by activated phagocytic cells abundantly present in periapical granulomas, can contribute to periapical tissue injury and bone loss in this disease.
We examined the oxidative burst (hydrogen peroxide-dependent oxidative product formation) of polymorphonuclear leukocytes (PMNL) in the peripheral blood from the patients with various types of periodontal diseases including localized juvenile (LJP), generalized juvenile (GJP) and adult periodontitis (AP). Heparinized peripheral blood was obtained from 15 LJP, 13 GJP and 52 AP patients and from 30 healthy control subjects. The oxidative product (2',7'-dichlorofluorescein; DCF) formation of PMNL by stimulation with phorbol myristate acetate myristate acetate was evaluated by a rapid quantitative assay using flow cytometry. The results indicated that all patient groups contained variable populations with normal or increased DCF formation, while the control subjects exhibited DCF formation as a single population. No significant differences in average DCF formation were found among the three patient groups. Although individual patients gave various values, the average DCF formation of the three patient groups was much higher than that of the control group. Statistical analysis revealed a significant positive correlation between DCF formation and the clinical periodontal parameters on an individual basis. Furthermore, after initial periodontal treatment, DCF formation decreased to normal levels. These results suggest that the capacity of peripheral blood PMNL to mount oxidative burst reactions might reflect the inflammatory status of periodontal disease.
A modification of Dische's carbazole reaction for uronic acid in the presence of borate is described. The advantages of the procedure are: 1.(1) There is an approximately twofold increase of sensitivity. The OD is a linear function of concentration between 4 and 40 μg/ml.2.(2) Maximum color develops immediately.3.(3) The color is stable for at least 16 hr.4.(4) There is greater reproducibility and reduction of interference by chloride ion and oxidants.It has been found possible to distinguish between heparin, heparin derivatives, and other polyuronides of connective tissue by comparing the effect of chlorides on the color yield in both procedures.