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Differential Gene Expression in Vascular Smooth Muscle Cells in Primary Atherosclerosis and In Stent Stenosis in Humans
Abstract
We sought to identify differentially expressed genes in human in stent stenosis (ISS) to provide insights into the mechanism of disease.
Using representation difference analysis, we examined differential gene expression between cultured normal human medial vascular smooth muscle cells (VSMCs) and cells from primary atherosclerotic plaques or ISS sites. Specific groups of genes were overexpressed in ISS and plaque VSMCs, including cell cycle regulatory proteins and cell matrix and contractile proteins. Differential expression was validated by virtual Northern analysis, reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. All ISS genes were expressed by normal intima and had even higher expression in primary plaque VSMCs.
ISS VSMCs have a stable gene expression profile reflecting an intimal pattern, intermediate between normal medial and primary plaque VSMCs. Differential expression profiling may identify markers of disease that are overexpressed in ISS and also help elucidate the origin of the ISS lesion.
... The result depicted that ERRFI1 gene expression in the control group (non-ISR) was higher than the case group (ISR) but despite the higher level of gene expression in the control group no significant differences have been observed between the two groups (p > 0.05). High expression of some genes has been reported in ISR (Zhang et al., 2002). ...
... We observed that the expression of the ERRFI1 gene was significantly higher in non-ISR individuals than ISR in diabetic patients (p < 0.05); however, in the non-diabetic group this difference was not statistically significant (p > 0.05). High expression of ERRFI1 as a stress-responsive protein can lead to cell cycle arrest, apoptosis of beta-cells, and ER stress in diabetes (Zhang et al., 2002). Induction of the ERRFI1 gene by cytokines, inhibits EGFR signaling in the beta cells. ...
Background
One of the common treatments in cardiovascular disease as the first cause of death in the world is stent implantation. In-Stent Restenosis (ISR) is the major drawback of stent implantation. As there are several lines of evidence suggesting genetic factors involved in ISR, in this study we evaluated the potential role of +808 T/G polymorphism in ERRFI1 and its quantitative expression in the development of ISR.
Material and methods
Individuals with ISR (n = 41) cases and Non-ISR (n = 51) controls, participated in this study. ERRFI1 gene expression in fresh un-stimulated PBMCs was determined in each group using quantitative real-time PCR. DNA extraction from the whole blood was performed using the phenol-chloroform method. The frequency of genotypes was determined by the PCR-RFLP technique.
Results
In spite of the higher amount of ERRFI1 expression in the control group (non-ISR), there were no statistically significant differences between ISR and non-ISR groups (p > 0.05). The expression of ERRFI1 was significantly different between ISR and Non-ISR groups only in patients with diabetes (p < 0.05). A significant association was observed between +808 T/G variant and ISR in patients with metabolic syndrome (p < 0.05).
Conclusions
The result of this study is suggesting that ERRFI1 gene expression may be associated with ISR in patients with diabetes. Therefore, ERRFI1 can be regarded as a target gene for therapeutic approaches. In addition, our data suggest a protective role for ERRFI1 + 808 T/G variant in the development of ISR in patients with metabolic syndrome.
... Abnormal proliferation, migration, and phenotypic switching of vascular smooth muscle cells (VSMCs) are key events in the pathogenesis of atherosclerosis and vascular restenosis. 1,2 VSMCs in normal vessels have remarkable plasticity that allows rapid adaptation to environmental cues through a switch from contractile to synthetic phenotype. 3 The contractile proteins, such as α-smooth muscle actin (α-SMA), transgelin/SM22α, heavy-caldesmon, calponin, and myosin heavy chain 11 (MYH11) are enriched in the quiescent but not proliferative VSMCs. ...
... 7,8 The expressions of both contractile markers and PDGFR are induced simultaneously during differentiation from MSCs to VSMCs and associated with the phenotypic switching and expansion of VSMCs in injured arteries of animal models. 2 Mechanistically, several transcriptional factors, such as Krüppel-like factors, class I bHLH factors, gp91phox-related NAD(P)H oxidase, serum response factor (SRF), myocardin, myocardin-related transcription factor B (MRTFb) and zinc finger homeobox 3, have been identified to transcriptionally regulate the expressions of VSMC contractile markers and PDGFR. [9][10][11] Tripartite motif-containing (TRIM) proteins belong to the tripartite motif family and contain one coiled-coil region and three zinc-binding domains including one RING, one B-box type 1, and one B-box type 2 domains. ...
Vascular smooth muscle cells (VSMCs) in the normal arterial media continually express contractile phenotypic markers which are reduced dramatically in response to injury. Tripartite motif‐containing proteins are a family of scaffold proteins shown to regulate gene silencing, cell growth, and differentiation. We here investigated the biological role of tripartite motif‐containing 28 (TRIM28) and tripartite motif‐containing 27 (TRIM27) in VSMCs. We observed that siRNA‐mediated knockdown of TRIM28 and TRIM27 inhibited platelet‐derived growth factor (PDGF)‐induced migration in human VSMCs. Both TRIM28 and TRIM27 can regulate serum response element activity and were required for maintaining the contractile gene expression in human VSMCs. At the same time, TRIM28 and TRIM27 knockdown reduced the expression of PDGF receptor‐β (PDGFRβ) and the phosphorylation of its downstream signaling components. Immunoprecipitation showed that TRIM28 formed complexes with TRIM27 through its N‐terminal RING‐B boxes‐Coiled‐Coil domain. Furthermore, TRIM28 and TRIM27 were shown to be upregulated and mediate the VSMC contractile marker gene and PDGFRβ expression in differentiating human bone marrow mesenchymal stem cells. In conclusion, we identified that TRIM28 and TRIM27 cooperatively maintain the endogenous expression of PDGFRβ and contractile phenotype of human VSMCs.
... Previous studies have reported that Brg1 acted as an indispensable chromatin modulator during the embryonic development of the cardiovascular system, and maintained a low expression level in cardiovascular tissues after adulthood. 9,27 Other studies 13,14,28 have reported that Brg1 was involved in the pathophysiologic processes of various cardiovascular diseases. Zhang and colleagues 28 reported that Brg1 induced VSMC apoptosis in atherosclerosis. ...
... 9,27 Other studies 13,14,28 have reported that Brg1 was involved in the pathophysiologic processes of various cardiovascular diseases. Zhang and colleagues 28 reported that Brg1 induced VSMC apoptosis in atherosclerosis. Our previous studies showed that Brg1 was involved in aortic diseases via inducing apoptosis of HASMCs. ...
Objective:
To elucidate the mechanisms of Brahma-related gene 1 (Brg1) involvement in the pathophysiologic processes of aortic dissection.
Methods:
Seventeen dissecting, 4 dilated, and 10 healthy human aorta samples were collected. Expression of Brg1 in the medium of aorta was evaluated by quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical staining, respectively. The regulation effect of Brg1 on proliferation and migration of human aortic smooth muscle cells (HASMCs) was analyzed in 3 ways: using cell counting, a migration chamber, and a wound scratch assay. A polymerase chain reaction array was used for screening potential target genes of Brg1. A chromatin immunoprecipitation assay was adopted for direct deoxyribonucleic acid-protein binding detection.
Results:
Expression levels of Brg1 were increased in aortic dissection and aortic dilation patients. In vitro results indicated that overexpression of Brg1 inhibited proliferation and migration of HASMCs. The candidate proliferation- and migration-related Brg1 target gene found was Ras-related associated with diabetes (RRAD), expression levels of which were enhanced in dissecting aortic specimens. The direct regulation effect of Brg1 on RRAD was verified by chromatin immunoprecipitation assay results. Furthermore, down-regulating RRAD significantly alleviated the suppression effects of Brg1 on proliferation and migration of HASMCs.
Conclusions:
Our study illustrated that Brg1 inhibited the proliferation and migration capacity of HASMCs, via the mechanism of direct up-regulation of RRAD, thus playing an important role in the pathophysiologic processes of aortic dissection.
... VSMC genes are transcriptionally regulated through complex interactions of transcription factors and conserved cis-regulatory elements (Alexander and Owens 2012). Moreover, the expression of VSMC-selective genes is not dependent on the regulation of individual genes but depends on interactions of multiple gene expression (Zhang et al. 2002). VSMCs can undergo a series of pathological and physiological changes in transcription related regulatory processes. ...
Atherosclerosis (AS), leading to gradual occlusion of the arterial lumen, refers to the accumulation of lipids and inflammatory debris in the arterial wall. Despite therapeutic advances over past decades including intervention or surgery, atherosclerosis is still the most common cause of cardiovascular diseases and the main mechanism of death and disability worldwide. Vascular smooth muscle cells (VSMCs) play an imperative role in the occurrence of atherosclerosis and throughout the whole stages. In the past, there was a lack of comprehensive understanding of VSMCs, but the development of identification technology, including in vivo single-cell sequencing technology and lineage tracing with the CreERT2-loxP system, suggests that VSMCs have remarkable plasticity and reevaluates well-established concepts about the contribution of VSMCs. Transcription factors, a kind of protein molecule that specifically recognizes and binds DNA upstream promoter regions or distal enhancer DNA elements, play a key role in the transcription initiation of the coding genes and are necessary for RNA polymerase to bind gene promoters. In this review, we highlight that, except for environmental factors, VSMC genes are transcriptionally regulated through complex interactions of multiple conserved cis-regulatory elements and transcription factors. In addition, through a series of transcription-related regulatory processes, VSMCs could undergo phenotypic transformation, proliferation, migration, calcification and apoptosis. Finally, enhancing or inhibiting transcription factors can regulate the development of atherosclerotic lesions, and the downstream molecular mechanism of transcriptional regulation has also been widely studied.
... Long after inflammation is resolved and the endothelium is repaired (chronic phase), these cells continue to secrete extracellular matrix (ECM), leading to the progressive narrowing of the lumen. secreted by activated EC, platelets and immune cells stimulate ECM production and secretion, and reduce the expression of the contractile VSMC markers smooth muscle alpha-actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), calponin, and smooth muscle 22 alpha (SM22α) (42)(43)(44). In addition, the "activated" VSMC themselves produce cytokines such as TNFα and MCP-1, leading to positive feedback cascades of enhanced VSMC migration and proliferation (42). ...
Arterial occlusive disease is the narrowing of the arteries via atherosclerotic plaque buildup. The major risk factors for arterial occlusive disease are age, high levels of cholesterol and triglycerides, diabetes, high blood pressure, and smoking. Arterial occlusive disease is the leading cause of death in Western countries. Patients who suffer from arterial occlusive disease develop peripheral arterial disease (PAD) when the narrowing affects limbs, stroke when the narrowing affects carotid arteries, and heart disease when the narrowing affects coronary arteries. When lifestyle interventions (exercise, diet…) fail, the only solution remains surgical endovascular and open revascularization. Unfortunately, these surgeries still suffer from high failure rates due to re-occlusive vascular wall adaptations, which is largely due to intimal hyperplasia (IH). IH develops in response to vessel injury, leading to inflammation, vascular smooth muscle cells dedifferentiation, migration, proliferation and secretion of extra-cellular matrix into the vessel’s innermost layer or intima. Re-occlusive IH lesions result in costly and complex recurrent end-organ ischemia, and often lead to loss of limb, brain function, or life. Despite decades of IH research, limited therapies are currently available. Hydrogen sulfide (H 2 S) is an endogenous gasotransmitter derived from cysteine metabolism. Although environmental exposure to exogenous high H 2 S is toxic, endogenous H 2 S has important vasorelaxant, cytoprotective and anti-inflammatory properties. Its vasculo-protective properties have attracted a remarkable amount of attention, especially its ability to inhibit IH. This review summarizes IH pathophysiology and treatment, and provides an overview of the potential clinical role of H 2 S to prevent IH and restenosis.
... Collectively, the mechanisms of VSMC phenotype switching and its effect on vascular remodeling are orchestrated by diverse transcription factors, and the mechanisms are still not fully understood . A large number of VSMC genes are transcriptionally regulated due to mitogenic stimulation, suggesting an upper level of transcriptional regulation that controls gene expression networks rather than individual genes (Zhang et al., 2002). Interestingly, emerging evidence has revealed that VSMCs undergo epigenetic alterations during phenotypic modulation and vascular remodeling, which provide such upper-level regulation of transcription . ...
Atherosclerosis is a chronic inflammatory disease characterized by extensive remodeling of medium and large-sized arteries. Inward remodeling (=lumen shrinkage) of the vascular walls is the underlying cause for ischemia in target organs. Therefore, inward remodeling can be considered the predominant feature of atherosclerotic pathology. Outward remodeling (=lumen enlargement) is a physiological response compensating for lumen shrinkage caused by neointimal hyperplasia, but as a pathological response to changes in blood flow, outward remodeling leads to substantial arterial wall thinning. Thinned vascular walls are prone to rupture, and subsequent thrombus formation accounts for the majority of acute cardiovascular events. Pathological remodeling is driven by inflammatory cells which induce vascular smooth muscle cells to switch from quiescent to a proliferative and migratory phenotype. After decades of intensive research, the molecular mechanisms of arterial remodeling are starting to unfold. In this mini-review, we summarize the current knowledge of the epigenetic and transcriptional regulation of vascular smooth muscle cell phenotype switching from the contractile to the synthetic phenotype involved in arterial remodeling and discuss potential therapeutic options.
... The generation of hpNFAT RNA dONs following AAV9 transduction was verified in 5 µm myocardial frozen sections by fluorescent in situ hybridization according to standard protocols [47]. A molecular beacon with complementary sequence to the dON was used as a probe (Biomers, Ulm, Germany). ...
Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.
... L'athérosclérose, est associée à des facteurs de risque physiopathologique ou environnementaux tels que l'hypertension, l'hyperlipidémie, le diabète, tabagisme, sédentarité, infections et stress. Par ailleurs, le territoire vasculaire et les conditions de flux modifient l'expression génique des artères (Zhang, Goddard et al. 2002). Elle affecte les courbures de tronc artériel ou les bifurcations (les artères coronaires, les artères crâniennes et extra-crâniennes, la crosse aortique, les artères fémorales et dans une moindre mesure, les artères rénales) ( Figure 15). ...
Les Microvésicules (MVs) sont des marqueurs circulants de l’activation cellulaire au cours de la dysfonction du greffon et l’athérothrombose. Les MVs de la cellule bêta ou intratissulaires vasculaires, sont peu connues. Le suivi longitudinal de 19 patients transplantés d’îlots pancréatiques par les MV- PSA-NCAM+ sanguines témoigne d’une altération précoce du greffon avant l’identification des marqueurs clinique et biologique de la perte du greffon. La cinétique de libération des MVs leucocytaires, endothéliales et hépatiques suggère leur intérêt pour l’identification de la cause de la perte du greffon et pour la surveillance de l'immunosuppression. Après validation d’une méthode d’extraction douce des MVs tissulaires sur des plaques d’athérome, nous avons mesuré l’accumulation de MVs pro-sénescentes dans l’aorte de rats âgés, qui étaient réduite par l’ingestion d’EPA : DHA (6 :1), avec une baisse des propriétés pro-sénescentes identifiée sur les cellules endothéliales d’artères coronaires en culture. Le contrôle des MV nocives par cytoprotecteur du vaisseau réduirait la sénescence endothéliale.
... These not terminally differentiated but mature cells retain phenotypic plasticity [8], pivotal for the vascular remodelling processes [9]. Exogenous and microenvironmental stimuli may induce SMC phenotypic switching, i.e. changes in the expression of SMC lineage-associated markers [10], and activation of signalling pathways. In vitro differentiation of SMC towards endothelial cells is reported [11], but transdifferentiation in human atherosclerotic lesions is still an object of debate [12]. ...
Background:
Vascular smooth muscle cells exhibit phenotypic plasticity in response to microenvironmental stimuli and contribute to vascular remodelling through mechanisms only partially understood. In atherosclerosis, P2X-purinoceptor7 (P2X7) has been related to interleukin-1β (IL-1β) and metalloproteinase 9 (MMP9). The hypoxia-inducible factor-1alpha (HIF1α) was associated to remodelling. Here the activation of IL-1β and MMP9 was studied in relationship to P2X7 and HIF1α in cells exploited from human carotid plaque and internal mammary artery.
Methods and results:
Migrating cells expressed HIF1α-regulated canopy FGF-signalling regulator 2 and CD117, and led to primary cells with SMC-like phenotype (VSMC), P2X7+. We investigated in VSMC the effects of hypoxia, of treatment with tumour necrosis factor-α (TNFα) and/or with P2X7 antagonist, A740003. Quantitative RT-PCR showed that hypoxia unaffected IL-1β and down-regulated MMP9 mRNAs, without activating HIF1α. TNFα increased IL-1β mRNA via NLR Family Pyrin Domain-Containing 3, with production of proIL-1β but no rise of mature IL-1β. Zymography demonstrated that A740003 triggered MMP9 secretion from VSMC. Combination of A740003 with TNFα abrogated this effect. Combination was ineffective on IL-1β activation elicited by TNFα, but down-regulated HIF1α mRNA. A740003 induced the intracellular P2X7 aggregation and differently perturbed lysosome and mitochondria network compared to TNFα.
Conclusions:
Cells migration from human arteries leads to partially differentiated VSMC analogous to neointimal cells within atherosclerotic lesions. Down-regulated HIF1α in stimulated VSMC translates in resilience in atherosclerotic lesions. P2X7-independent partial activation of IL-1β elicited by TNFα underlines complexity of the cytokine secretion. Data also supported P2X7 as modulator of MMP9 secretion, important for atherosclerosis progression.
... However, the effects of TGFβ on SMC behaviour are more variable, with studies showing that TGFβ can both inhibit and stimulate SMC proliferation and migration. This may be due to the heterogeneous nature of SMCs, as evidenced by the varying gene expression patterns of human SMCs derived from primary atherosclerotic plaques, in-stent stenoses or healthy arteries [133]. At the molecular level, these differences have been attributed to varying levels of receptor expression, membrane localisation of receptors, availability of intracellular signalling mediators and presence of transcriptional co-regulators within the nucleus (reviewed in [54,134]). ...
Excessive vascular smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis are key events in the development of intimal hyperplasia, a pathophysiological response to acute or chronic sources of vascular damage that can lead to occlusive narrowing of the vessel lumen. Atherosclerosis, the primary cause of coronary artery disease, is characterised by chronic vascular inflammation and dyslipidemia, while revascularisation surgeries such as coronary stenting and bypass grafting represent acute forms of vascular injury. Gene knockouts of transforming growth factor-beta (TGFβ), its receptors and downstream signalling proteins have demonstrated the importance of this pleiotropic cytokine during vasculogenesis and in the maintenance of vascular homeostasis. Dysregulated TGFβ signalling is a hallmark of many vascular diseases, and has been associated with the induction of pathological vascular cell phenotypes, fibrosis and ECM remodelling. Here we present an overview of TGFβ signalling in SMCs, highlighting the ways in which this multifaceted cytokine regulates SMC behaviour and phenotype in cardiovascular diseases driven by intimal hyperplasia.
... It is clear that VSMCs phenotypic switching is characterized by changes in gene expression patterns that underlie the changes of cell functional properties. The transformation from contractile phenotype to synthetic phenotype is associated with the silencing of differentiation marker genes expression and the upregulation of genes that facilitate other cellular functions such as cell proliferation or migration [7][8][9]. The transcription factor regulates genes expression by binding to response elements in the promoter regions of target genes in kinds of tissues including VSMCs. ...
Abnormal phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of cardiovascular diseases such as atherosclerosis, hypertension and restenosis after angioplasty. Transcription factors have emerged as critical regulators for VSMCs function, and recently we verified inhibiting transcription factor Gax was important for controlling VSMCs proliferation and migration. This study aimed to determine its role in phenotypic modulation of VSMCs. Western blot revealed that overexpression of Gax increased expression of VSMCs differentiation marker genes such as calponin and SM-MHC 11. Then, Gax overexpression potently suppressed proliferation and migration of VSMCs with or without platelet-derived growth factor-induced-BB (PDGF-BB) stimuli whereas Gax silencing inhibited these processes. Furthermore, cDNA array analysis indicated that Rap1A gene was the downstream target of Gax in human VSMCs. And overexpression of Gax significantly inhibited expression of Rap1A in VSMCs with or without PDGF-BB stimuli. Moreover, overexpression of Rap1A decreased expression of VSMCs differentiation marker genes and increased proliferation and migration of VSMCs with or without PDGF-BB stimuli. Finally, Gax overexpression significantly inhibited the neointimal formation in carotid artery injury of mouse models, specifically through maintaining VSMCs contractile phenotype by decreasing Rap1A expression. In conclusion, these results indicated that Gax was a regulator of human VSMCs phenotypic modulation by targeting Rap1A gene, which suggested that targeting Gax or its downstream targets in human VSMCs may provide an attractive approach for the prevention and treatment of cardiovascular diseases.
... Based on available gene expression microarray data of HaoSMCs, we performed an educated guess siRNA screen targeting 2000 genes which have been described to be differentially expressed in the phenotypic switch, mitosis, apoptosis and cell migration [22][23][24]. An siRNA library targeting these 2000 genes was acquired from the extended druggable Silencer® siRNA Libraries (Lifetech, Ambion, Austin, TX, USA). 3 different siRNAs per gene were arranged in 21, 384-well plates. ...
Purpose:
Understanding of the mechanisms of vascular smooth muscle cells (VSMCs) phenotypic regulation is critically important to identify novel candidates for future therapeutic intervention. While HTS approaches have recently been used to identify novel regulators in many cell lines, such as cancer cells and hematopoietic stem cells, no studies have so far systematically investigated the effect of gene inactivation on VSMCs with respect to cell survival and growth response.
Methods and results:
257 out of 2000 genes tested resulted in an inhibition of cell proliferation in HaoSMCs. After pathway analysis, 38 significant genes were selected for further study. 23 genes were confirmed to inhibit proliferation, and 13 genes found to induce apoptosis in the synthetic phenotype. 11 genes led to an aberrant nuclear phenotype indicating a central role in cell mitosis. 4 genes affected the cell migration in synthetic HaoSMCs. Using computational biological network analysis, 11 genes were identified to have an indirect or direct interaction with the Osteopontin pathway. For 10 of those genes, levels of proteins downstream of the Osteopontin pathway were found to be down-regulated, using RNAi methodology.
Conclusions:
A phenotypic high-throughput siRNA screen could be applied to identify genes relevant for the cell biology of HaoSMCs. Novel genes were identified which play a role in proliferation, apoptosis, mitosis and migration of HaoSMCs. These may represent potential drug candidates in the future.
... Additionally, higher levels of TM2 and CAPG were found in atherosclerotic tissue. α-Tropomyosin is overexpressed in plaque VSMCs [39] and CAPG has been found augmented in conditions of increased motility in stimulated VSMCs by hypoxia [40]. ...
Background:
Cardiovascular disease (CVD) is the leading cause of death globally, being atherosclerosis the main cause. Main risk factors are known and current effort is very much dedicated to improve prevention. However, the asymptomatic and silent course of atherosclerosis hampers an accurate and individualized risk evaluation.
Objectives:
Here we investigate subjacent molecular changes taking place in arterial tissue which can be ultimately translated in a measurable fingerprint in plasma.
Methods:
First, we applied a combined approach to find out main molecular alterations at protein and metabolite level in response to early atherosclerosis development in a rabbit model. A potential reflection of all these alterations observed in aortic tissue was investigated in rabbit plasma and further analyzed in a translational study in human plasma from 62 individuals.
Results:
Data link the structural remodeling taking place in atherosclerotic arteries in terms of loss of contractile properties and favored cellular migration, with an up-regulation of integrin linked kinase, tropomyosin isoform 2 and capping protein gelsolin-like, and a down-regulation of vinculin. A molecular response to oxidative stress is evidenced, involving changes in the glucose metabolism enzymes pyruvate kinase (PKM) and phosphoglycerate kinase (PGK), and pyruvate. Up-regulation of aspartate connects different changes observed in amino acids metabolism and, additionally, alterations in the phosphatydilcholine route of the glycerophospholipid metabolism were found.
Conclusions:
A specific molecular marker panel composed by PKM, valine and pyruvate is shown here linked to cardiovascular risk.
... Gene expression studies have identified many genes or proteins to be either up-or down-regulated in animal atherosclerosis models (3,4) or human atherosclerosis plaques (5)(6)(7). However, the genes reported mainly focus on aspects of regulation of cell apoptosis (8)(9)(10), growth (11), mobility (12), inflammation (13), and signal transduction (14,15), while the mechanisms of lipid droplet (LD) formation were largely ignored or underestimated in these studies. ...
A large number of macrophage-derived foam cells stores excessive neutral lipids in intracellular droplets, and plays a major role during the development of atherosclerosis. The formation and catabolism of intracellular lipid droplets (LDs) are regulated by LD-associated proteins, a group of proteins which are located on the surface of LDs and regulate the formation, morphology and lipolysis of LDs. In order to illustrate the function of LD-associated proteins during the process of atherosclerosis, the foam cell model is induced by oxidized low-density lipoprotein (ox-LDL) in macrophages originated from the THP-1 cell line, and cDNA microarrays are used to monitor the gene expression profiles of LD-associated proteins. Gene expression data show that 2% of changed genes are lipid binding genes during the transformation of foam cells. The major candidate genes, the cell death-inducing DFF45-like effector (CIDE) family and Perilipin, Adipophilin, and TIP47 (PAT) family, have different alterations during the formation of foam cells. CIDEB, CIDEC, Adipophilin, S3-12 and LSDP5 were up-regulated, while TIP47 was down-regulated. There was no significant change in CIDEA and Perilipin. These results were confirmed by real-time PCR and immunoblotting. This study presents a comprehensive analysis of the gene expression of LD-associated proteins during the differentiation of human foam cells, which may play an important role in the process of atherosclerosis.
... Previous studies showed that BRG1 overexpression could significantly increase the apoptotic rate of mesenchymal stem cells and some tumor cells [19,20]. Recently, Zhang et al. showed that BRG1 was upregulated in vascular SMC of patients with primary atherosclerosis and was involved in the pathophysiological process of atherosclerosis presumably by inducing cell apoptosis [32]. In the present study, we found that BRG1 is overexpressed in aortic SMC and its level was positively correlated with the percentage of apoptotic aortic SMCs in the aortic media of TAD. ...
Background:
Here we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD).
Methods:
BRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and alpha-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis.
Results:
The expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells.
Conclusions:
BRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.
... Related studies show that BRG1 overexpression can significantly increase the apoptotic rate of mesenchymal stem cells [17]. Similarly, a study by Zhang et al. [18] showed that BRG1 is up-regulated in VSMCs of patients with primary atherosclerosis and is involved in the pathophysiological process of atherosclerosis, presumably by inducing cell apoptosis. In our in vitro assay, our results suggest that BRG1 overexpression can promote cell apoptosis and reduce cell proliferation in VSMCs. ...
OBJECTIVES Brahma-related gene 1 (BRG1) and long non-coding RNAs (lncRNAs) play important roles in cellular processes. However, little
is known regarding their roles in thoracic aortic aneurysms. We investigated BRG1 expression in thoracic aortic aneurysms
and the roles of BRG1 and the lncRNA HIF 1 alpha-antisense RNA 1 in regulating the proliferation and apoptosis of aortic smooth
muscle cells in vitro.
... VSMC proliferation, the hallmark of vascular proliferative diseases, involves multiple genes that are transcriptionally regulated in response to injury or mitogenic signals implicating a higher level of regulation exists in VSMC that controls entire gene expression program required for proliferation [7,8]. This regulation involves epigenetic mechanisms, which by altering the chromatin structure and dynamics control transcriptional gene activation that are essential for normal development and maintenance of organisms and to facilitate their interaction with surrounding environment [3,4,[9][10][11]. ...
The histone deacetylase (HDAC) inhibitors, butyrate and trichostatin A (TSA), are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC) that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi) by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.
... Our data herein suggest that acute exposure to apo(a) is chemorepulsive to human SMC of both arterial and venous origin although the mechanism of action is undoubtedly complex and involves multiple signalling pathways, including integrin V 3. It is feasible that the magnitude of apo(a) effect could be governed by differential expression and activation of various signalling pathways in particular subsets (phenotypes) of SMC within vascular walls. The validity of such a hypothesis is upheld by studies in which SMC diversity in atherosclerotic plaques and restenotic lesions has been demonstrated ( Adams et al., 2006;Zhang et al., 2002). ...
Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVβ3-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVβ3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.
... Proteomics analyses in carotid tissue have generated biomarkers for cardiovascular events, such as osteopontin [5]. Only transcriptomic approaches have so far been applied to SMC from human atherosclerotic lesions [6,7], while no study has thus far compared the proteome of SMC derived from symptomatic or asymptomatic carotid disease and control vascular tissue. ...
Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.
... In models of atherosclerosis and in-stent restenosis, BRG1 in particular has been found to be up-regulated in SMCs in vivo. 87 Further, SMC marker gene induction in the murine heart by LIM-only CRP2 (a SMC differentiation co-factor) appears to be dependent on its binding to BRG1. 88 BRG1 or BRM appears to be critical for SMC-specific gene activation by MRTFA. ...
Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.
... Thus, clonality is more likely to be explained by the presence of developmental clones in the normal vessel wall, rather than a mutation. Finally, in contrast to tumours, plaque VSMCs show poor proliferation, enhanced apoptosis, and early senescence [50]. These features would not confer a proliferative or survival advantage to plaque VSMCs. ...
Atherosclerosis is the main cause of coronary heart disease and stroke, the two major causes of death in developed society. There is emerging evidence of excess risk of cardiovascular disease at low radiation doses in various occupationally exposed groups receiving small daily radiation doses. Assuming that they are causal, the mechanisms for effects of chronic fractionated radiation exposures on cardiovascular disease are unclear. We outline a spatial reaction-diffusion model for atherosclerosis and perform stability analysis, based wherever possible on human data. We show that a predicted consequence of multiple small radiation doses is to cause mean chemo-attractant (MCP-1) concentration to increase linearly with cumulative dose. The main driver for the increase in MCP-1 is monocyte death, and consequent reduction in MCP-1 degradation. The radiation-induced risks predicted by the model are quantitatively consistent with those observed in a number of occupationally-exposed groups. The changes in equilibrium MCP-1 concentrations with low density lipoprotein cholesterol concentration are also consistent with experimental and epidemiologic data. This proposed mechanism would be experimentally testable. If true, it also has substantive implications for radiological protection, which at present does not take cardiovascular disease into account. The Japanese A-bomb survivor data implies that cardiovascular disease and cancer mortality contribute similarly to radiogenic risk. The major uncertainty in assessing the low-dose risk of cardiovascular disease is the shape of the dose response relationship, which is unclear in the Japanese data. The analysis of the present paper suggests that linear extrapolation would be appropriate for this endpoint.
Arterial occlusive disease is the leading cause of death in Western countries. Core contemporary therapies for this disease include angioplasties, stents, endarterectomies and bypass surgery. However, these treatments suffer from high failure rates due to re-occlusive vascular wall adaptations and restenosis. Restenosis following vascular surgery is largely due to intimal hyperplasia. Intimal hyperplasia develops in response to vessel injury, leading to inflammation, vascular smooth muscle cells dedifferentiation, migration, proliferation and secretion of extra-cellular matrix into the vessel’s innermost layer or intima. In this review, we describe the current state of knowledge on the origin and mechanisms underlying the dysregulated proliferation of vascular smooth muscle cells in intimal hyperplasia, and we present the new avenues of research targeting VSMC phenotype and proliferation.
Aims
Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome.
Methods and results
Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.
Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs.
Conclusion
This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.
MicroRNAs have emerged as critical regulators in the pathogenesis of asthma. However, the role of microRNAs in asthma needs to be further elucidated. In this study, we found that miR-139-5p was greatly decreased in airway smooth muscle (ASM) cells from asthmatic humans as well as ASM cells stimulated with cytokines. Overexpression of miR-139-5p markedly suppressed ASM cell proliferation and promoted cell apoptosis, whereas knockdown of miR-139-5p had the opposite effect. Further study verified that Brg1, a chromatin remodeling factor, was upregulated in ASM cells treated with cytokines and acted as a direct target of miR-139-5p. Ectopic expression of Brg1 partially reversed the effect of miR-139-5p on cell proliferation and apoptosis. Moreover, overexpression of Brg1 restored miR-139-5p-induced downregulation of Akt and p70S6 K phosphorylation. Together, these data indicate that miR-139-5p may function as a key regulator of ASM cell proliferation and apoptosis, potentially by targeting the Brg1 gene, and thus suggesting a potential role of miR-139-5p in the pathogenesis of asthma.
More than 50 years ago, William Kannel1 first popularized the concept of cardiovascular disease risk factors for clinicians and clinical epidemiologists.2 The word “factor” derives from Latin (meaning doer) which implies causality.
Ca(2+) is an important second messenger in vascular smooth muscle cells (VSMCs). Therefore VSMCs exercise tight control of intracellular Ca(2+) concentration ([Ca(2+) ]i ) by expressing a wide repertoire of Ca(2+) channels and transporters. The presence of several pathways for Ca(2+) influx and efflux provides many possibilities for controlling [Ca(2+) ]i in a spatial and temporal manner. [Ca(2+) ]i has a dual role in VSMCs; firstly it is necessary for VSMC contraction and, secondly, it can activate multiple transcription factors. These factors are cAMP response element binding protein (CREB), nuclear factor of activated T lymphocytes (NFAT) and serum response factor (SRF). Furthermore it was recently reported that the C-terminus of voltage-dependent L-type Ca(2+) calcium channels can regulate transcription in VSMCs. Transcription regulation in VSMCs modulates gene expression patterns; including genes coding for contractile and cytoskeleton proteins and those promoting proliferation and cell growth. Dependent on their gene expression VSMCs can exist in different functional states or phenotypes. The majority of healthy VSMCs display a contractile phenotype, characterized by a high contractile ability and a low proliferative rate. However VSMCs can undergo phenotypic modulation with different physiological and pathological stimuli whereby they start to proliferate, migrate and synthesize excessive extracellular matrix. These events are associated with injury repair and angiogenesis but also with development of cardiovascular pathologies such as atherosclerosis and hypertension. This review discusses the currently known Ca(2+) - dependent transcription factors in VSMCs, their regulation by Ca(2+) signalling and their role in VSMCs phenotype. This article is protected by copyright. All rights reserved.
Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.
A central goal of molecular cardiology is the characterization of molecular mechanisms regulating complex cardiovascular functions
and the identification of diseaseassociated gene-expression patterns. Gene transcription is the most important regulatory
mechanism by which a phenotype and functional state of a cell and a tissue is determined. Therefore, qualitative and quantitative
assessment is the first step into the nature of biological processes. When small cell numbers are used, messenger RNA (mRNA)
abundance is easier to investigate in a comprehensive manner than protein expression.
Carotid artery stenosis is known to be a significant risk factor for stroke and indeed extracranial atheroma is the single
most important contributor to non-haemorrhagic stroke in the developed world. Studies such as the European Carotid Surgery
Trial (ECST) [63], [11] and North American Symptomatic Carotid Endarterectomy Trial (NASCET) [25], despite some methodological differences, demonstrated a significant benefit for endarterectomy in symptomatic carotid stenosis
of more than 70%. More recently, evidence has been published in the form of the Asymptomatic Carotid Surgery Trial (ACST)
[34], that intervention in the asymptomatic population with a stenosis of 70% or more may be of benefit providing the associated
risks and likely morbidity of the intervention is low.
To estimate the effects of forced repeated swimming stress on BNP serum levels in rats.
Adult male Wistar rats weighting between 280-330 g were divided into two groups: control group (n = 8) and stress group (n = 8). Rats in the stress group were exposed to forced swimming stress daily, for 7 days. The rats were forced to swim in plastic tanks (90 cm wide, 120 cm deep) containing tap water (temperature ca. 25 degrees C). The depth of water was 40 cm. Duration of each swimming session progressively increased from 10 minutes on the first day to 40 minutes on days 6 and 7. Rats were sacrificed and blood was drawn from abdominal aorta for BNP analysis immediately after the last swimming session. B-type natriuretic serum level was determined by ELISA method using RAT BNP-32 kit (Phoenix Pharmaceutical Inc.).
There was no statistically significant difference between mean BNP serum level in the stress group after the swimming period (0.81 +/- 0.14 ng/ml) as compared to the unstressed group of rats (0.8 +/- 0.08 ng/ml). After the swimming period mean body weight slightly decreased in the stress group in comparison with values before stress period (296.3 g vs. 272.8 g), but this difference was not statistically significant. The stress period had no influence on food intake in the stress rat group.
The workload consisting of 40-minutes long swimming session is not sufficient to provoke BNP release from myocardium in rats.
To analyze usefulness of measurement amino-terminal pro-B type natriuretic peptide of (NT pro-BNP) as the one of parameters of water overload in patients with chronic kidney diseases.
A total number of 277 patients with chronic kidney diseases (CKD) were followed up in the period often years between January 2000 and July 2010. Patients with creatinine clearance of 60 ml/min or less were included in the study. Changes of creatinine clearance, and in last five years changes of NT pro-BNP were followed. Water overload was analyzed using chest x-ray in relation with concentration of NT pro-BNP in the blood.
Decrease of clearance of creatinine ranged from average 54.7 ml/min in the first year to 14.6 ml/min in the fifth year of the monitoring. Average NT pro-BNP level in patients without any sign of water overload was 94 pg/ml (SD 21), mean value in those with Kerley lines was 231 pg/ml/L (SD 64), in those with clear signs of water overload but without pleural effusion it was 525 pg/ml (SD 223), and in those with water retention including pleural effusion it was 1606 pg/ml (SD 1134). Using test of multiple correlation a statistically significant correlation between X-ray signs of water overload and NT pro-BNP concentration was shown, p < 0.05.
Measurement of NT pro-BNP was increased in the beginning of water overload in patients with CKD. Increased value of NT pro-BNP may be found earlier than any other signs of water overload. NT pro-BNP was a useful parameter in estimation of water overload in these patients.
To investigate the presence and expression levels of the IL-18 in the primary breast cancer tissue in relation to the unchanged breast tissue in same patients and the breast tissue in patients with benign breast disease, as well as the correlation between the IL-18 expression levels and pathohistological factors, including the correlation between IL-18 expression and the estrogens and progesterone receptor status.
This prospective randomized study was conducted at the Policlinic for Laboratory Diagnostics of the University Clinical Centre of Tuzla. 50 patients with invasive ductal breast cancer and 20 patients with benign breast diseases were included in the study. The tree-step immunohistochemical staining was used for testing the levels of IL-18 expression and hormone receptor status.
IL-18 was present in the breast cancer tumour, in the surrounding unchanged tissue of the same patients and in the breast tissue of patients with benign breast tumour and other benign breast disease. The expression of this interleukin was significantly higher in breast cancer tumour tissue as compared to its expression in surrounding unchanged tissue of the same patients (p < 0.05), whereas IL-18 expression was not significantly higher in breast cancer tumours compared to its expression in breast tissue of the patients with benign breast diseases (p = 0.057). There was no significant correlation between IL-18 expression and the lymph node status, and between IL-18 expression and the pathohistological factors.
The results suggest possible involvement of IL-18 in complex mechanisms of breast carcinogenesis.
To investigate the cystatin C levels in sera of patients with aggressive non-Hodgkin B-cell lymphoma.
The levels of cystatin C in sera of lymphoma patients and control group consisted of healthy individuals, were measured by using specific sandwich-type ELISA. For each patient the clinical stage of disease was determined according to Ann Arbor staging system for lymphomas.
Our study shows that mean cystatin C serum level in the patients group (1056 +/- 65 ng/mL) was significantly higher when compared with the mean level of the healthy control group (819 +/- 28 ng/mL) (P = 0.001). Mean cystatin C level of the group with clinical stages III and IV (1255 +/- 109 ng/mL) was significantly elevated when compared with the mean level of the group with clinical stages I and II (896 +/- 51 ng/mL) (P = 0.03).
This finding points out a connection between inhibitor level and aggressive behaviour of lymphoma and could be considered for further strategies of prognosis of the disease.
To investigate the hypothesis that in patients with coronary atherosclerosis it is possible to measure plasma mRNA levels from genes responsible for plaque development and destabilization.
Methods for RNA isolation, mRNA transcription and quantitative PCR were evaluated and optimised, in order to achieve reliable mRNA quantification. RESULTS mRNA level was possible to quantify from plasma of patients with coronary atherosclerosis, as well as from healthy volunteers, from genes encoding cathepsin S, cathepsin B, CD40 molecule, monocyte chemotactic protein 1, death-associated protein kinase 1, matrix metallopeptidase 9, vascular cell adhesion molecule 1 and phosphoglycerate kinase 1 (reference gene). Analytical between-run imprecision of average threshold cycle, expressed as coefficient of variation was below 2%. EDTA blood samples should be centrifuged within one hour of venesection. It was not possible to quantify plasma mRNA level from genes encoding macrophage scavenger receptor 1, perilipin, tissue factor, phospholipase A2 group IIA, collagen type I alpha 2 and interleukin 1 alpha.
Further plasma mRNA analysis is reasonable to access its potential usefulness in non-invasive in vivo monitoring of gene expression profile in vascular beds.
Differences in the frequency of distribution of the cytochrome P450 (CYP) allelic variants have been demonstrated between distinct ethnic groups, contributing to observed interindividual variation in drug response. In this study we determined, for the first time, prevalence of the common allelic variants of the polymorphic CYP enzymes, CYP3A4*1B and CYP3A5*3, in the population of Bosnia and Herzegovina (BH).
Genomic DNA was extracted from blood samples collected from 140 unrelated subjects. A real-time PCR was used for the detection of CYP polymorphisms, with the application of the specific TaqMan SNP Genotyping Assay (Applied Biosystems) for CYP3A5*3, while CYP3A4*1B was genotyped by high-resolution melting analysis.
Our results have shown that the distribution of CYP3A4*1B and CYP3A5*3 alleles was in line with the data reported in European Caucasians. We confirmed that CYP3A4*1B mutant allele is rare in Caucasians, being present in only 5.1% individuals. However, CYP3A5*3 polymorphism was found to be predominant in the Bosnian population with an incidence of 94%, similarly to other European populations tested so far. Interestingly, we have demonstrated a strong linkage disequilibrium between CYP3A5*3 and CYP3A4*1B alleles. No significant difference in allele frequencies for CYP3A4*1B and CYP3A5*3 has been shown between male and female subjects participating in our study.
Our data demonstrated the high prevalence of CYP3A5*3 allele in Bosnian population, indicating significance of analysis of CYP3A5 and CYP3A4 polymorphisms and corresponding allele frequencies in specific ethnic groups. Importantly, results of this study may lead to translation of pharmacogenetics and individualized therapeutic approach in current clinical practices in BH.
Lipin 1 is a recently discovered multifunctional protein involved in the metabolism of lipids, while PPARgamma is involved in adipocyte differentiation, and regulation of lipid metabolism. Up to now, LPIN1 and PPARG gene polymorphisms have been associated with type 2 diabetes, metabolic syndrome, and central obesity. In this study, we hypothesized that genetic variants within LPIN1 and PPARG genes were associated with traits of metabolic syndrome. Correlation between biochemical parameters (including but not limited to, glucose, HbA1c, insulin levels, HDL and LDL cholesterol, triglycerides, serum proteins, liver enzymes) and frequency of polymorphisms in LPIN1 (rs11693809 and rs2716610) and PPARG gene (rs10865710, rs3856806 and rs1801282), was tested in this study.
The study included 70 patients diagnosed with metabolic syndrome and type 2 diabetes. Two polymorphisms of LPIN1 gene (rs11693809 and rs2716610), and three polymorphisms of PPARG gene (rs10865710, rs385806 and rs1801282) were analyzed by real time PCR and conventional PCR-RFLP methods.
Our analysis revealed correlation between insulin levels and rs11693809 LPIN1 polymorphism in diabetic patients. Also the results of this study showed an association of rs10865710 and rs385806 polymorphism of PPARG with HDL cholesterol and LDL plus total cholesterol levels, respectively.
These data reflect an association of analyzed PPARG and LPIN1 gene polymorphisms with values of insulin, HDL, LDL and total cholesterol witch indicates an important role of these genes in lipid metabolism and pathogenesis of type 2 diabetes and metabolic syndrome.
The study presents a case of endometrial cancer in a breast cancer patient treated with tamoxifen. The disease occured with elevated values of CA 125 and CA 15_3 tumour markers without any other signs. Additional diagnostic analyses were performed showing a "de novo" endometrial cancer rather than metastatic breast cancer. The patient underwent surgery and radiotherapy. Thereafter, the values of tumour markers were in the reference values.
Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive.
In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA-mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G(1) phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip). Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury.
These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.
Aim To analyze usefulness of measurement amino-terminal pro-B type natriuretic peptide of (NT pro-BNP) as the one of parameters of water overload in patients with chronic kidney diseases. Mmethods A total number of 277 patients with chronic kidney diseases(CKD) were followed up in the period of ten years between January 2000 and July 2010. Patients with creatinine clearance of 60 ml/min or less were included in the study. Changes of creatinine clearance, and in last ive years changes of NT pro-BNP were followed. Water overload was analyzed using chest x-ray in relation with concentration of NT pro-BNP in the blood. Results Decrease of clearance of creatinine ranged from average 54,7 ml/min in the irst year to 14,6 ml/min in the ifth year of the monitoring. Average NT pro-BNP level in patients without any sign of water overload was 94 pg/ml (SD 21), mean value in thosewith Kerley lines was 231 pg/ml/L (SD 64), in those with clear signs of water overload but without pleural effusion it was 525 pg/ml (SD 223), and in those with water retention including pleural effusion it was 1606 pg/ml (SD 1134). Using test of multiple correlation a statistically signiicant correlation between X-ray signs of water overload and NT pro-BNP concentration was shown, p<0,05. Conclusion Measurement of NT pro-BNP was increased in the beginning of water overload in patients with CKD. Increased value of NT pro-BNP may be found earlier than any other signs of water overload. NT pro-BNP was a useful parameter in estimation of water overload in these patients.
A large number of macrophage-derived foam cells stores excessive neutral lipids in intracellular droplets, and plays a major role during the development of atherosclerosis. The formation and catabolism of intracellular lipid droplets (LDs) are regulated by LD-associated proteins, a group of proteins which are located on the surface of LDs and regulate the formation, morphology and lipolysis of LDs. In order to illustrate the function of LD-associated proteins during the process of atherosclerosis, the foam cell model is induced by oxidized low-density lipoprotein (ox-LDL) in macrophages originated from the THP-1 cell line, and cDNA microarrays are used to monitor the gene expression profiles of LD-associated proteins. Gene expression data show that 2% of changed genes are lipid binding genes during the transformation of foam cells. The major candidate genes, the cell death-inducing DFF45-like effector (CIDE) family and Perilipin, Adipophilin, and TIP47 (PAT) family, have different alterations during the formation of foam cells. CIDEB, CIDEC, Adipophilin, S3-12 and LSDP5 were up-regulated, while TIP47 was down-regulated. There was no significant change in CIDEA and Perilipin. These results were confirmed by real-time PCR and immunoblotting. This study presents a comprehensive analysis of the gene expression of LD-associated proteins during the differentiation of human foam cells, which may play an important role in the process of atherosclerosis.
Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses. The molecular mechanisms are unknown. TGF-beta is known to be involved in the fibroproliferative responses of vascular smooth muscle cells (VSMCs) in restenosis, and TGF-beta antagonists attenuate ISR. We hypothesized that SS ions induce the latent TGF-beta activator, thrombospondin-1 (TSP1), through altered oxidative signaling to stimulate increased TGF-beta activation and VSMC phenotype change.
VSMCs were treated with SS metal ion cocktails, and morphology, TSP1, extracellular matrix production, desmin and TGF-beta activity were assessed by immunoblotting.
SS ions stimulate the synthetic phenotype, increased TGF-beta activity, TSP1, increased extracellular matrix and downregulation of desmin in VSMCs. Furthermore, SS ions increase hydrogen peroxide and decrease cGMP-dependent protein kinase (PKG) signaling, a known repressor of TSP1 transcription. Catalase blocks SS ion attenuation of PKG signaling and increased TSP1 expression.
These data suggest that ions from stent alloy corrosion contribute to ISR through stimulation of TSP1-dependent TGF-beta activation.
Full-text of this article is not available in this e-prints service. This article was originally published following peer-review in Pathobiology, published by and copyright Karger. Formation of unstable plaques frequently results in atherothrombosis, the major cause for ischaemic stroke, myocardial infarction and peripheral arterial disease. Patients who have symptomatic thrombosis in one vascular bed are at increased risk of disease in other beds. However, the development of the disease in carotid, coronary and peripheral arteries may have different pathophysiology suggesting that more complex treatment protocols may have to be designed to reduce plaque development at different locations. In this review we describe the known risk factors, compare the developmental features of coronary and carotid plaque development and determine their association with end-point ischaemic events. Differences are also seen in the genetic contribution to plaque development as well as in the deregulation of gene and protein expression and cellular signal transduction activity of active cells in regions susceptible to thrombosis. Differences between carotid and coronary artery plaque development might help to explain the differences in anatomopathological appearance and risk of rupture.
The origin of vascular smooth muscle cells that accumulate in the neointima in vascular diseases such as transplant arteriosclerosis, atherosclerosis and restenosis remains subject to much debate. Smooth muscle cells are a highly heterogeneous cell population with different characteristics and markers, and distinct phenotypes in physiological and pathological conditions. Several studies have reported a role for bone marrow-derived progenitor cells in vascular maintenance and repair. Moreover, bone marrow-derived smooth muscle progenitor cells have been detected in human atherosclerotic tissue as well as in in vivo mouse models of vascular disease. However, it is not clear whether smooth muscle progenitor cells can be regarded as a 'friend' or 'foe' in neointima formation. In this review we will discuss the heterogeneity of smooth muscle cells, the role of smooth muscle progenitor cells in vascular disease, potential mechanisms that could regulate smooth muscle progenitor cell contribution and the implications this may have on designing novel therapeutic tools to prevent development and progression of vascular disease.
See article by Martinet et al. [19] in this issue .
Cardiovascular diseases (CVDs) have a long track record, reaching pandemic proportions with the industrialization of the world. Current estimates of the World Health Organization project that, in less than 7 years from now, CVD will become the leading cause of death in developing countries and that, in less than 13 years from now, 25 million people will die from CVD on a global scale each year [1]. A major proportion of these CVD deaths will be related to atherosclerotic CVD (ASCVD) and, particularly, to its complication stage [2]. Hence, there is a strong call to the cardiovascular research community to identify pivotal mediators of atherosclerotic lesion formation and complication which at some point may turn into therapeutic targets.
What may be considered as searching for a needle in a haystack has been tremendously facilitated by the availability of the microarray technique. This technique, which allows the detection of labeled RNA hybridizing to DNA molecules attached to a solid surface, evolved in a number of steps in the 1960s and 1970s and was finally moved to high capacity in the 1990s [3,4]. Microarray plates with a density of >250,000 oligonucleotides or >10,000 cDNA per cm2 currently serve as a versatile screening probe in genomics research, and it will not be long until arrays become available that allow the screening of the entire genome on one chip for less than one cell and 1 μg of tissue [5,6]. With regard to cardiovascular medicine, DNA microarray assays have already identified a three-digit number of genes potentially involved in atherosclerosis [7–16].
As marvelous as these assays are, they are not without concern. Indeed, a number of potential pitfalls to these kinds of genomic studies have been reported, …
*Tel.: +1-507-255-1296; fax: +1-507-255-1824. Email address: herrmann.joerg{at}mayo.edu
Carotid endarterectomy (CEA) has been used for the past several decades in patients with carotid occlusive disease. Large randomized controlled trials have documented that CEA is a highly effective stroke preventive among patients with carotid stenosis and recent transient ischemic attack or cerebral infarction. In asymptomatic patients with carotid stenosis, clinical trial data suggest that the degree of stroke prevention from CEA is less than among symptomatic patients. However, otherwise healthy men and women with an asymptomatic carotid stenosis of 60% or greater have a lower risk of future cerebral infarction, including disabling cerebral infarction, if treated with CEA compared with those treated with medical management alone. More recently, carotid artery stenting has been performed Increasingly for patients with carotid occlusive disease. As technology has improved, procedural risks have declined and are approaching those reported for CEA. The benefits and durability of CEA compared with carotid artery stenting are still unclear and are being studied in ongoing randomized controlled trials.
In this study we investigated the morphology of grafts from the internal thoracic artery and the great saphenous vein, before their use in aortocoronary bypass surgery, in order to draw conclusions concerning their suitability and viability.
Sections of grafts from the great saphenous vein and left internal thoracic artery obtained for use in bypass surgery were examined using light microscopy and transmission and scanning electron microscopy.
The histological changes in the walls of the vessels were classified as acute or chronic. The acute lesions concerned the endothelium and the subendothelial layer. There was extensive necrosis of endothelial cells, resulting in the basement membrane being left uncovered and becoming the target of blood cells. The endothelial necrosis was accompanied by subendothelial oedema and focal destruction of the inner elastic lamina of the internal thoracic artery. The chronic lesions affected mostly the venous grafts and included the presence of distinct atheromatous plaques or thickening of the intima and media.
The combination of ischaemic and chronic atheromatous lesions in bypass grafts may contribute to a decrease in their viability, especially in the case of venous grafts.
In dividing cells, the RNA-binding protein HuR associates with and stabilizes labile mRNAs encoding proliferative proteins, events that are linked to the increased cytoplasmic presence of HuR. Here, assessment of HuR levels in various vascular pathologies (intimal hyperplasia, atherosclerosis and neointimal proliferation, sclerosis of arterialized saphenous venous graft, and fibromuscular dysplasia) revealed a distinct increase in HuR expression and cytoplasmic abundance within the intima and neointima layers. On the basis of these observations, we postulated a role for HuR in promoting the proliferation of vascular smooth muscle cells. To test this hypothesis directly, we investigated the expression, subcellular localization, and proliferative influence of HuR in human vascular smooth muscle cells (hVSMCs). Treatment of hVSMCs with platelet-derived growth factor increased HuR levels in the cytoplasm, thereby influencing the expression of metabolic, proliferative, and structural genes. Importantly, knockdown of HuR expression by using RNA interference caused a reduction of hVSMC proliferation, both basally and following platelet-derived growth factor treatment. We propose that HuR contributes to regulating hVSMC growth and homeostasis in pathologies associated with vascular smooth muscle proliferation.
Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.
Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative
splicing. In the case of the α-tropomyosin (α-TM) gene, exon 2a rather than exon 2b is specifically spliced in α-TM-SM mRNA,
which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of α-tropomyosin
and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular
cloning and Western and Northern blottings have revealed that in addition to the down-regulation of β-TM-SM, α-TM-SM converted
to α-TM-F1 and α-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in
culture. Simultaneously, a change of caldesmon isoforms from high M
r type to low M
r type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast,
cultured smooth muscle cells maintaining a differentiated phenotype continued to express α-TM-SM, β-TM-SM, and high M
r caldesmon. In situhybridization revealed specific coexpression of α-TM-SM and highM
r caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent
expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.
Proteoglycans are important constituents of blood vessels and accumulate in various forms of vascular disease. Little is known concerning the proteoglycan composition of restenotic lesions formed after angioplasty and whether the proteoglycan composition of these lesions differs from that of primary atherosclerosis. Accordingly, we sought to characterize the distribution of two proteoglycans, biglycan and decorin, in primary atherosclerotic and restenotic lesions of human coronary arteries. Restenosis (n = 37) and primary (n = 11) lesions obtained from 48 patients by directional atherectomy of human coronary arteries were stained with antibodies against biglycan and decorin. To further characterize the extracellular matrix of restenotic tissues, we studied the co-distribution of these proteoglycans with collagen types I, III, and IV. The loose fibroproliferative tissue seen predominantly in restenosis lesions consistently stained positively for biglycan in patterns of deposition ranging from disseminated to homogeneous. The density and intensity of biglycan staining was correlated with the density of collagen type I and III fiber networks, both of which were observed to interweave among the loose fibroproliferative tissue. The compact connective tissue of primary atherosclerotic plaque was characterized by strong biglycan staining which co-localized with intense collagen type I and III staining. Only basement membrane-like structures rich in collagen type IV demonstrated negative biglycan staining. In contrast, loose fibroproliferative tissue exhibited no significant staining for decorin. Strong immunostaining for decorin, however, was found in primary atherosclerotic plaque. There are thus regional differences in the distribution of extracellular matrix proteoglycans of restenotic and primary human atherosclerotic lesions; these observations suggest that differences established for the biological roles of biglycan and decorin in other organ systems may extend as well to pathologically altered human coronary arteries.
Restenosis due to neointima formation is the major limitation of stent-supported balloon angioplasty. Despite abundant animal data, molecular mechanisms of neointima formation have been investigated on only a limited basis in patients. This study sought to establish a method for profiling gene expression in human in-stent neointima and to identify differentially expressed genes that may serve as novel therapeutic targets.
We retrieved tissue specimens from patients with symptomatic in-stent restenosis using a novel helix cutter atherectomy device. cDNA samples prepared from neointima (n=10) and, as a control, from the media of normal arteries (n=14) were amplified using a novel polymerase chain reaction protocol and hybridized to cDNA arrays. Immunohistochemistry characterized the atherectomy material as neointima. cDNA arrays readily identified differentially expressed genes. Some of the differentially expressed genes complied with expected gene expression patterns of neointima, including downregulation of desmin and upregulation of thrombospondin-1, cyclooxygenase-1, and the 70-kDa heat shock protein B. Additionally, we discovered previously unknown gene expression patterns, such as downregulation of mammary-derived growth inhibitor and upregulation of FK506-binding protein 12 (FKBP12). Upregulation of FKBP12 was confirmed at the protein level in neointimal smooth muscle cells.
Gene expression patterns of human neointima retrieved by helix-cutter atherectomy can be reliably analyzed by cDNA array technology. This technique can identify therapeutic targets in patients, as exemplified by the findings regarding FKBP12. FKBP12 is the receptor for Rapamycin (sirolimus), which in animal models reduced neointima formation. Our study thus yields a rationale for the use of Rapamycin to prevent restenosis in patients.
Apoptosis (programmed cell death) of VSMCs is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously shown that plaque-derived VSMCs are intrinsically sensitive to apoptosis, irrespective of the local plaque microenvironment. The mechanism of this effect is unclear, although we have also shown that IGF-1 is a potent survival factor for human VSMCs, and plaque VSMCs are very sensitive to apoptosis induced by p53, which acts in part through suppression of IGF-1 signalling. We therefore analysed immortalised human coronary plaque and normal medial VSMCs by ligand binding, Western blots, and assays of IGF-1 targets that mediate protection against apoptosis, such as the serine/threonine kinase akt. Coronary plaque VSMCs showed markedly reduced IGF-1 binding (100%) than medial VSMCs (178±30%), indicating lower surface expression of the IGF- I receptor (IGF-IR). This was confirmed with Western blots which showed a reduced total expression of IGF-IR on plaque VSMCs compared with normal medial VSMCs. Plaque also VSMCs had an impaired akt response to IGF-I (five-fold lower akt response at 2min than normal medial VSMCs), and impaired protection against apoptosis by exogenous IGF-1 (3.5 fold lower response). We conclude that human plaque VSMCs show an intrinsic sensitivity to apoptosis due to defective expression of IGF-1 receptor, and impaired IGF-1-mediated survival signalling. Thus impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.
Vascular tissue obtained using a directional percutaneous atherectomy device was examined microscopically. Tissue was obtained from coronary arteries without prior instrumentation (primary lesions, n = 31), aortocoronary saphenous vein bypass grafts with primary lesions (n = 8), coronary arteries with lesions developing after prior balloon angioplasty or mechanical atherectomy (restenotic lesions, n = 30) and vein bypass grafts with restenotic lesions (n = 4). Primary lesions were characterized by dense intimal fibrosis with necrotic debris (83% of intimal tissue) and foam cells typical of atherosclerosis. These lesions frequently contained cholesterol crystals (45% of coronary arteries, 50% of vein grafts) and calcium deposits (65% of coronary arteries, 38% of vein grafts). Restenotic lesions were characterized by an increased proportion of loose fibroproliferative tissue (45% of coronary artery intima, 35% of vein graft intima). Immunohistochemical stains confirmed this proliferative tissue to be primarily smooth muscle cells. Thrombus was rarely observed. Comparison of resected tissues indicated that dense fibrosis and necrosis are significantly more common in primary than in restenotic lesions (83% versus 56% of intimal tissue, p = 0.0005), whereas smooth muscle cell hyperplasia is more common in restenotic than in primary lesions (44% versus 17% of intimal tissue, p less than 0.0005). Partial-thickness resection of medial tissue or full-thickness resection of media with associated adventitial tissue occurred in 27 (56%) of 39 primary atheromatous lesions and 16 (47%) of 34 restenotic lesions; subintimal tissue obtained from primary lesions appeared identical to that obtained from restenotic lesions. These data indicate that the histopathologic characteristics of the neointimal layer of restenotic lesions differ from those of the intimal layer of primary atherosclerotic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in gene expression underlie many biological phenomena, including cellular differentiation and activation, embryonic development, and pathological processes. In recent years, much attention has been focused on the identification of genes that are differentially expressed between normal and disease states: the isolation of disease-specific genes is not only essential for our understanding of the molecular basis of pathological conditions, but may potentially highlight novel therapeutic targets.
Cell size distribution and growth rates were studied in vitro in human plaque cells from advanced primary stenosing and fresh restenosing lesions of peripheral and coronary arteries. Cells were isolated either by the explant technique or by enzymatic disaggregation and were identified as smooth muscle cells by their typical growth pattern and their positive reaction with antibodies against smooth muscle alpha-actin. Endothelial cells were found in plaque specimens from coronary arteries but were only present in primary cultures. Smooth muscle cells from primary stenosing tissue (ps-SMC) exhibited a significantly lower growth rate in culture (0.15 +/- 0.04 population doublings per day; means +/- SD) compared with cells from restenosing lesions (re-SMC; 0.60 +/- 0.13 population doublings per day; means +/- SD). ps-SMC usually became senescent in their second passage, i.e., after 5-7 cumultive population doublings. re-SMC retained their high proliferative activity even after five passages (15 cumulative population doublings). Cell populations of both origins consisted of two distinct subpopulations which could be discriminated by cell size measurements: relatively small, predominant cells (cell diameter: 18.0 +/- 4 microns; means +/- SD) and large fibroblast-like cells (cell diameter: 26.0 +/- 3 microns; means +/- SD). The proportion of large cells was higher in cell populations derived from primary stenosing tissue. These results suggest that stenosing plaque tissue from human peripheral and coronary arteries consists of two smooth muscle cell subpopulations. The low proliferative activity of total smooth muscle cell populations of advanced primary stenosing lesions contrasts with the high mitotic activity of smooth muscle cells obtained from secondary stenosing intimal proliferates.
To further understand the temporal mode and mechanisms of coronary restenosis, 229 patients were studied by prospective angiographic follow-up on day 1 and at 1, 3 and 6 months and 1 year after successful percutaneous transluminal coronary angioplasty. Quantitative measurement of coronary stenosis was achieved by cinevideodensitometric analysis. Actuarial restenosis rate was 12.7% at 1 month, 43.0% at 3 months, 49.4% at 6 months and 52.5% at 1 year. In 219 patients followed up for greater than or equal to 3 months, mean stenosis diameter was 1.91 +/- 0.53 mm immediately after coronary angioplasty, 1.72 +/- 0.52 mm on day 1, 1.86 +/- 0.58 mm at 1 month and 1.43 +/- 0.67 mm at 3 months. In 149 patients followed up for greater than or equal to 6 months, mean stenosis diameter was 1.66 +/- 0.58 mm at 3 months and 1.66 +/- 0.62 mm at 6 months. In 73 patients followed up for 1 year, mean stenosis diameter was 1.65 +/- 0.56 mm at 6 months and 1.66 +/- 0.57 mm at 1 year. Thus, stenosis diameter decreased markedly between 1 month and 3 months after coronary angioplasty and reached a plateau thereafter. In conclusion, restenosis is most prevalent between 1 and 3 months and rarely occurs beyond 3 months after coronary angioplasty.
This study represents a systematic analysis of the fine-structural characteristics of atherosclerotic lesions of the superficial femoral artery in man together with the growth characteristics in culture of the smooth muscle cells derived from these lesions. Occlusive fibrous atherosclerotic plaques were obtained from 29 male patients at the time of bypass surgery for occlusion of the superficial femoral artery and were studied by light and transmission electron microscopy. The occluded segment of each artery was obtained immediately after removal from the patient and examined with sterile techniques, and representative segments were fixed for light- and electron-microscopic study. Adjacent segments were used for dissection of the lesion away from the underlying media, and smooth muscle cells were cultured from lesion and nonlesion areas and compared in terms of their growth responses to increasing concentrations of a pool of human whole blood serum. The majority of the lesions were fibroproliferative and contained relatively little lipid. The fibrous cap that covered each lesion consisted of a special form of dense connective tissue that contained flat, pancake-shaped smooth muscle cells in a lacunalike space. This space consisted of concentric layers of basement membrane, collagen fibrils, and proteoglycan. The majority of the cells beneath the fibrous cap were smooth muscle cells mixed with small but varying numbers of macrophages. Most of the lesions were occluded by a thrombus, which had undergone organization and recanalization. A small number of the lesions had deep lipid deposits together with foci of degeneration and calcification. The occluded thrombi contained smooth muscle cells and a larger proportion of macrophages than the lesions themselves. The in vitro growth properties of the smooth muscle cells isolated from the lesion and the underlying media suggested that the lesion cells had senesced, compared with the medial smooth muscle cells derived from the same artery.
We studied death of human vascular smooth muscle cells derived from coronary plaques and normal coronary arteries and aorta. Cells from normal arteries underwent death only upon removal of serum growth factors. In contrast, plaque-derived cells died even in high serum conditions, and death increased after serum withdrawal. Death was characteristically by apoptosis in both normal and plaque-derived cells, as determined by time-lapse videomicroscopy, electron microscopy, and DNA fragmentation patterns. IGF-1 and PDGF were identified as potent survival factors in serum, whereas EGF and basic fibroblast growth factor had little effect. Stable expression of bcl-2, a protooncogene that regulates apoptosis in other cell lines, protected smooth muscle cells from apoptosis, although there was no detectable difference in endogenous bcl-2 expression between cells from plaques or normal vessels. We conclude that apoptosis of human vascular smooth muscle cells is regulated by both specific gene products and local cytokines acting as survival factors. Apoptosis may therefore regulate cell mass in the normal arterial wall and the higher rates of apoptosis seen in plaque smooth muscle cells may ultimately contribute to plaque rupture and breakdown and thus to the clinical sequelae of atherosclerosis.
Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome
these problems, the PCR-coupled subtractlve process of representational difference analysis (RDA) [Lisitsyn,N. et al. (1993)
Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating
V(D)J recombination, were identified in a genomic transfectant by cDNA RDA In a small fraction of the time taken by conventional
means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes.
Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated
clones were obtained from the pre-B cell line 1-8, Including IGF-1B, and a predicted homologue of the natural killer cell
antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives.
cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single
gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses,
and examination of pathogen - host interactions.
The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro.
Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of extracellular matrix proteins. The applicability to humans of experimental animal models of these processes has been questioned.
Primary atherosclerotic and restenotic lesions were excised by percutaneous directional coronary atherectomy in 93 patients. Smooth muscle cells were cultivated by an explant technique and identified by their morphology in culture, ultrastructural features under electron microscopy and immunostaining using monoclonal antibodies to smooth muscle cell alpha-actin. Proliferation in secondary culture was assessed with growth curves and the synthesis of collagen and sulfated glycosaminoglycans by the incorporation of 3H-proline and 35S-sulfate, respectively. These studies were also performed in cells derived from human umbilical artery media.
Success rates for primary (45%) and secondary (12%) culture of coronary cells were not influenced by clinical variables or lesion category. Primary culture success was improved by the presence of organized thrombus in the plaque and in relation to increased maximal cell density of the atherectomy specimen. Restenotic cells displayed more rapid growth than did cells of primary atherosclerotic origin, which grew in a manner similar to that of umbilical artery cells. Mean calculated population-doubling times for the three cell groups were 52 h (95% confidence interval [CI] 48 to 58 h), 71 h (95% CI 62 to 83 h) and 74 h (95% CI 65 to 84 h), respectively. Restenotic and primary atherosclerotic cells did not differ in the synthesis of collagen ([mean +/- SEM] 0.034 +/- 0.004 vs. 0.033 +/- 0.004 nmol isotope.microgram protein-1, p = NS) or sulfated glycosaminoglycans (11.47 +/- 1.07 vs. 15.37 +/- 3.10 nmol isotope.microgram protein-1, p = NS), but the coronary cells synthesized significantly more collagen and sulfated glycosaminoglycans than did umbilical artery cells (0.019 +/- 0.004 and 5.43 +/- 1.00 nmol isotope.microgram protein-1, respectively, both p < 0.05).
These data indicate that increased smooth muscle cell proliferation contributes to coronary restenosis in humans and support the concept that the extracellular matrix synthesis of adult smooth muscle cells is important to lesion formation.
To isolate specific markers of both differentiated and proliferating vascular smooth muscle cells (VSMCs), we used the technique of differential cDNA screening using RNA from cultured rat aortic VSMCs. The tissue specificity of expression of all of the cDNAs isolated was determined by Northern analysis. We isolated seven distinct cDNAs that were more strongly expressed in freshly dispersed, differentiated, aortic VSMCs compared with dedifferentiated late-passage cells. These were the cDNAs for tropoelastin, a matrix protein; alpha-smooth muscle (SM) actin, gamma-SM actin, calponin, and phospholamban, which are all proteins associated with the contractile function of differentiated VSMCs; SM22 alpha, a smooth muscle-specific protein of unknown function, and CHIP28, a putative membrane channel protein that is not highly expressed in other SM tissues and may therefore be a new VSMC marker. Two cDNAs that were expressed preferentially in late-passage dedifferentiated VSMCs were also isolated. These were the cDNAs for osteopontin and matrix Gla protein (MGP). Like CHIP28, MGP was strongly expressed in aortic VSMCs but not in other types of tissues containing SM cells, suggesting that both have specific functions in vascular tissue. Osteopontin and MGP have both previously been isolated from developing bone. Their expression in proliferating VSMCs suggests that they may be involved in regulating the calcification that commonly occurs in vascular lesions. The set of cDNAs obtained extends the range of DNA probes that are available for identifying VSMCs and characterizing their phenotype in vivo by in situ hybridization. Therefore, they should aid in the analysis of gene expression during the development of vessel lesions.
We used intravascular ultrasound (IVUS) imaging to evaluate the chronic vessel responses to Palmaz-Schatz stents.
Palmaz-Schatz stents have been shown to inhibit early elastic recoil and late arterial remodeling while triggering neointimal hyperplasia. However, changes occurring in native vessels surrounding stent struts have not been well studied.
Postintervention and follow-up (mean [+/-SD] 5.4 +/- 3.8 months) serial IVUS imaging was performed in 25 stents without restenosis and 24 with in-stent restenosis. Intravascular ultrasound imaging using automatic transducer pullback at 0.5 mm/s allowed measurement at 1-mm axial increments of external elastic membrane (EEM), stent and lumen cross-sectional areas (CSAs) and calculation of peristent plaque plus media (P + M = EEM - stent) CSA, intrastent plaque (stent-lumen) CSA, arterial remodeling (delta EEM CSA), tissue growth outside the stent (delta P + M CSA) and tissue growth within the stent (delta stent-lumen CSA). Volumes were calculated using the Simpson rule.
Mean EEM CSA increased significantly from 16.9 +/- 5.0 mm2 after intervention to 18.4 +/- 4.9 mm2 at follow-up (p < 0.0001), reflecting an increase in P + M CSA surrounding the stent (1.6 +/- 1.3 mm2). Greater tissue growth within the stent (2.4 +/- 2.2 mm2) correlated weakly, but directly with tissue growth surrounding the stent (r = 0.356, p = 0.0121). The ratio of peristent/intrastent tissue growth correlated weakly with arterial remodeling (r = 0.282, p = 0.0525). Restenotic stents had more tissue growth both within and surrounding the stent than did nonrestenotic stents. Volumetric measurements, which could be obtained in 15 lesions, showed similar results.
After implantation there is a chronic increase in plaque mass both within and surrounding the stents. The increase in peristent plaque mass is associated with adaptive remodeling.
To clarify the mechanisms of restenosis, restenotic human tissue specimens obtained by directional coronary atherectomy (DCA) in 43 patients were immunohistochemically analysed for cell proliferation and deposition of PG-M/versican, an important extracellular matrix proteoglycan of the vessel wall. The patients were classified into five groups according to the period after percutaneous transluminal coronary angioplasty (PTCA): 0-1 month (N = 6), 1-3 months (N = 12), 3-6 months (N = 11), more than 6 months (N = 6) and de novo lesions (N = 8). The tissue specimens were of 35 restenotic lesions following PTCA and eight primary stenotic lesions with no prior PTCA. Total cell numbers in the atherectomy specimens increased significantly up to 3 months after PTCA. Most cells were alpha-smooth muscle actin (alpha-SMA)-positive. To evaluate cell proliferation, the specimens were immunostained for Ki-67 antigen (clone MIB-1). A significant increase in the positive ratio was observed up to 1 month after PTCA, although the labelling index was less than 1 per cent at every stage. The deposition of PG-M/versican, as analysed by immunohistochemistry, was greatest during the period 1-3 months after primary angioplasty, when restenosis detected by angiography progresses most actively. These results suggest that the peak of cell proliferation in the neointima occurs earlier than angiographic restenosis and that the deposition of PG-M/versican may be a major factor in restenosis following angioplasty.
Representational difference analysis (RDA) is a powerful technique for cloning the differences between genomes, and has recently
been adapted for cloning differentially expressed genes. RDA, like other PCR-based differential screening methods, is prone
to the production of false positives. We have identified a major source of false positives in RDA of cDNA and have introduced
improvements which minimise their production. These modifications also significantly increase sensitivity, allowing for the
isolation of rare differential transcripts from nanogram amounts of mRNA.
Several studies have shown that single-dose administration of agents that inhibit medial cell replication, such as antisense oligonucleotides to cell replication genes, can inhibit neointima formation after arterial injury. However, the precise mechanism of action of these agents is unknown. We analyzed the effect of phosphorothioated oligonucleotides delivered periadventitially on the response to injury in the balloon-injured rat carotid artery. Antisense oligonucleotides to c-myc suppressed medial replication 2 days after injury, but this effect was not present at 4 or 14 days. Endothelial cell proliferation was not affected by antisense oligonucleotides. There was, however, a significant suppression of intimal area and intima/media ratio at 14 days and an increase in lumen area in the antisense-treated group. Indeed, an increase in the number of medial cells at 14 days in the antisense group indicated that most of the effect of the agent was due to the suppression that most of the effect of the agent was due to the suppression of cell migration. No effect was noted on expression of two genes, osteopontin and tropoelastin, used as markers of modulation of smooth muscle cells to a "neonatal" phenotype at 4 days after injury. Because no effect on cell proliferation could be demonstrated after 2 days, our data indicate that an early effect of the antisense agent mediates its longer-term effects. We suggest that this effect may be due to the suppression of migration of medial smooth muscle cells rather than the suppression of medial or intimal cell proliferation.
Compared with vascular smooth muscle cells (VSMCs) from normal vessels, VSMCs from human atherosclerotic plaques proliferate more slowly, undergo earlier senescence, and demonstrate higher levels of apoptosis in culture. The tumor suppressor genes p105RB (retinoblastoma, acting through the E2F transcription factor family) and p53 regulate cell proliferation, cell senescence, and apoptosis in many cell types. We have therefore determined whether these stable growth properties of plaque VSMCs reflect altered activity of RB and/or p53. VSMCs were derived from coronary atherectomies or from normal coronary arteries from transplant recipients. Compared with normal VSMCs, plaque VSMCs showed a higher ratio of the active (hypophosphorylated) to the inactive (phosphorylated) form of RB and a lower level of E2F transcriptional activity. Cells were stably transfected with retrovirus constructs that inhibited RB or p53 alone or in combination. Suppression of RB alone increased rates of cell proliferation and apoptosis and inhibited cell senescence in normal VSMCs. Suppression of p53 and RB together had similar effects but, additionally, resulted in immortalization of normal VSMC cultures. In contrast, inhibition of RB binding to E2F or ectopic expression of E2F-1 in plaque VSMCs induced massive apoptosis, which required suppression of p53 to rescue cells. Suppression of RB and p53 together increased cell proliferation and delayed senescence but failed to immortalize plaque VSMCs. Inhibition of p53 alone had minimal effects on plaque VSMCs but increased the lifespan of normal VSMCs. We conclude that human plaque VSMCs have slower rates of cell proliferation and earlier senescence than do cells from normal vessels because of a defect in phosphorylation of RB. Furthermore, both disruption of RB/E2F and inhibition of p53 are required for plaque VSMCs to proliferate without apoptosis. This observation may explain the relatively low level of cell proliferation and high level of apoptosis seen in VSMCs in human atherosclerotic plaques.
SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling. We have identified an interaction
between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required
for G1/S transition. BRG1 and BAF155, mammalian homologs of yeast SWI2 and SWI3, respectively, are found in cyclin E complexes and
are phosphorylated by cyclin E-associated kinase activity. In this report, we show that overexpression of BRG1 causes growth
arrest and induction of senescence-associated β-galactosidase activity, which can be overcome by cyclin E. Our results suggest
that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive
state.
We used an antisense oligonucleotide (ODN) to inhibit laminin (LM) beta1 chain synthesis in mouse embryonic lung explants and cell cultures. The ODN spanned 17 bases located 13 bases downstream the initiation codon and contained phosphorothioate and C-5 propynyl pyrimidine modifications. Penetration of the ODN into the lung explants was confirmed by fluorescein isothiocyanate (FITC) tagging. 50 microM of antisense ODN decreased LM beta1 chain synthesis by 82+/-6.9% with no significant changes in the synthesis of other LM chains. The same antisense probe but without C-5 propynyl pyrimidine modification, another 17-mer ODN complementary to the LM beta1 initiation codon, and a 17-mer ODN complementary to the LM alpha1 initiation codon had no antisense activity. Lung explants exposed to the active LM beta1 antisense ODN showed decreased LM-1 and collagen type IV deposition at the epithelial-mesenchymal interface and an arrest in bronchial smooth muscle (SM) development. Histological examination and cell motility assays suggested that this arrest was due to impaired spreading and migration of SM cell precursors over the defective basement membrane (BM). Our studies indicate that beta1-chain containing LMs play a role in bronchial myogenesis.
We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, approximately 35% of currently named human genes (approximately 11,000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5+/-12.6 (mean+/-SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0+/-1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3+/-0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.
Proliferation and alpha(v)beta(3) integrin-dependent migration of vascular smooth muscle cells are suppressed on polymerized type I collagen. To identify genes specifically regulated in human smooth muscle cells by polymerized collagen, we used the suppressive subtraction hybridization technique. Compared with smooth muscle cells cultured on monomer collagen, polymerized collagen suppresses the following: (1) a number of other extracellular matrix proteins, including fibronectin, thrombospondin-1, tenascin-C, and cysteine-rich protein 61; (2) actin binding proteins including alpha-actinin; (3) signaling molecules; (4) protein synthesis-associated proteins; and (5) genes with unknown functions. Some of the identified genes, including cysteine-rich protein 61, show unique kinetics of mRNA regulation by monomer or polymerized collagen distinct from growth factors, suggesting extracellular matrix-specific gene modulation. Moreover, in vivo balloon catheter-mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen. Protein levels of thrombospondin-1 and fibronectin are also suppressed by polymerized collagen. Thrombospondin-1-mediated smooth muscle cell migration on vitronectin is significantly inhibited after culture on polymerized collagen for 24 hours, which is associated with decreased alpha-actinin accumulation at focal adhesions. Thus, polymerized type I collagen dynamically regulates gene expression, pericellular accumulation of extracellular matrix molecules, and the response to a given matrix molecule.
Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque-derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% (125)I-IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase-mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1-mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.
The most effective immediate cure for coronary stenosis is stent-supported angioplasty. Restenosis due to neointima proliferation represents a major limitation. We investigated the expression of 2435 genes in atherectomy specimens and blood cells of patients with restenosis, normal coronary artery specimens, and cultured human smooth muscle cells (SMCs). Of the 223 differentially expressed genes, 37 genes indicated activation of interferon-gamma (IFN-gamma) signaling in neointimal SMCs. In cultured SMCs, IFN-gamma inhibited apoptosis. Genetic disruption of IFN-gamma signaling in a mouse model of restenosis significantly reduced the vascular proliferative response. Our data suggest an important role of IFN-gamma in the control of neointima proliferation.
Although rupture of an atherosclerotic plaque is the major cause of acute vascular occlusion, the exact molecular mechanisms underlying this process are still poorly understood. In this study, we used suppression subtractive hybridization to make an inventory of genes that are differentially expressed in whole-mount human stable and ruptured plaques. Two libraries were generated, one containing 3000 clones upregulated and one containing 2000 clones downregulated in ruptured plaques. Macroarray analysis of 500 randomly chosen clones showed differential expression of 45 clones. Among the 25 clones that showed at least a 2-fold difference in expression was the gene of perilipin, upregulated in ruptured plaques, and the genes coding for fibronectin and immunoglobulin lambda chain, which were downregulated in ruptured plaques. Reverse transcriptase-polymerase chain reaction analysis on 10 individual ruptured and 10 individual stable plaques showed a striking consistency of expression for the clones SSH6, present in 8 ruptured and 2 stable plaques, and perilipin, expressed in 8 ruptured plaques and completely absent in stable plaques. Localization studies of both perilipin mRNA and protein revealed expression in cells surrounding the cholesterol clefts and in foam cells of ruptured atherosclerotic plaques. No expression was observed in nondiseased artery, and only a few cells in the shoulder region of stable plaques tested positive for perilipin. In conclusion, this study shows that it is possible to identify genes that are differentially expressed in whole-mount stable or ruptured atherosclerotic plaques. This approach may yield several potential regulators of plaque destabilization.
Restenosis after angioplasty or stenting remains the major limitation of both procedures. A vast array of drug therapies has been used to prevent restenosis, but they have proven to be predominantly unsuccessful. Recent trends in drug therapy have attempted to refine the molecular and biological targets of therapy, based on the assumption that a single biological process or molecule is critical to restenosis. In contrast, both stenting and brachytherapy, which are highly nonspecific, can successfully reduce restenosis after angioplasty or stenting, respectively. This review examines the biology of both angioplasty and stent stenosis, focussing on human studies. We also review the landmark human trials that have definitively proven successful therapies, such as stenting and brachytherapy. We suggest that the successful trials of stenting and brachytherapy and the failure of other treatments have highlighted the shortcomings of conventional animal models of arterial intervention, and gaps in our knowledge of human disease. In contrast to arguments advocating gene therapy, these studies suggest that the most likely successful drug therapy will have a wide therapeutic range, targeting as many of the components or biological processes contributing to restenosis as possible.