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Expression and function of angiotensin converting enzyme, chymase, and angiotensin II in the human radial artery and internal thoracic artery
Abstract
Background:
The potential role of the local renin-angiotensin system to differentially affect radial artery and internal thoracic artery graft performance has not been examined.
Methods:
Contractile responses to angiotensin I and II in the radial artery and the internal thoracic artery were examined in vitro. The expression function, and localization of angiotensin receptors, angiotensin converting enzyme, and chymase were studied in radial artery and internal thoracic artery segments.
Results:
Angiotensin I and II contractions were significantly greater (p < 0.05) in the radial artery compared to the internal thoracic artery. In both arteries, angiotensin II responses were mediated via the AT1 receptor. Messenger RNA transcripts for angiotensin-converting enzyme and chymase were detected in both arteries. Angiotensin-converting enzyme was localized to luminal and vaso vasorum endothelial cells and smooth muscle cells in both vessels, while chymase was colocalized with mast cells in adventitial and medial layers. An angiotensin converting enzyme or a chymase inhibitor singularly had no effect on angiotensin I contractions, however, when combined, a marked inhibition of the angiotensin I response was observed in both vessels.
Conclusions:
Our results illustrate the complexities which exist within the local renin angiotensin system and suggest that clinical trials which may modulate the system are warranted.
... Thus, in the human saphenous vein, mast cell chymase is present mainly in the adventitial layer of the vessel, with only a few cells staining positive for chymase in the media [8]. Similarly, in human radial and internal thoracic arteries, chymase is co-localised with mast cells in the adventitia, and occasionally in the medial layer of the radial artery [9]. The implication of this is that intravascular Ang I is more accessible to ACE than chymase. ...
... In other studies on isolated human blood vessels, ACE inhibitors or chymase inhibitors had no effect alone, but the combination reduced the responses to Ang I (saphenous vein [8], radial and internal thoracic artery [9], resistance vessels from subcutaneous gluteal fat biopsy [14]). Similar findings were obtained using resistance vessels from patients with coronary heart disease; the responses to Ang I were not altered by either enalaprilat or chymostatin alone, but were inhibited by the combination [31]. ...
Chymase is a chymotrypsin-like serine protease secreted from mast cells. Mammalian chymases are classified into two subgroups (alpha and beta) according to structure and substrate specificity; human chymase is an alpha-chymase. An important action of chymase is the ACE-independent conversion of Ang I to Ang II, but chymase also degrades the extracellular matrix, activates TGF-beta1 and IL-1beta, forms 31-amino acid endothelins and is involved in lipid metabolism. Under physiological conditions, the role of chymase in blood vessels is uncertain. In pathological situations, however, chymase may be important. In animal models of hypertension and atherosclerosis, chymase may be involved in lipid deposition and intimal and smooth muscle hyperplasia, at least in some vessels. In addition, chymase has pro-angiogenic properties. In human diseased blood vessels (e.g. atherosclerotic and aneurysmal aorta; remodeled pulmonary blood vessels), there are increases in chymase-containing mast cells and/or in chymase-dependent conversion of Ang I to Ang II. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of vascular disease. The effects of chymase can theoretically be attenuated either by reducing availability of the enzyme, with a mast cell stabiliser, or alternatively with specific chymase inhibitors. The mast cell stabiliser, tranilast, was shown to be beneficial in animal models of atherosclerosis, where a prevention protocol was used, but was not effective in clinical trials where it was administered after angioplasty. Chymase inhibitors could have the advantage of being effective even if used after injury. Several orally active inhibitors, including SUN-C8257, BCEAB, NK3201 and TEI-E548, are now available. These have yet to be tested in humans, but promising results have been obtained in animal models of atherosclerosis and angiogenesis. It is concluded that orally active inhibitors of chymase may have a place in the treatment of vascular diseases where injury-induced mast cell degranulation contributes to the pathology.
... Systemic hormones include the renin-angiotensinaldosterone system triggered by hypovolemia. Angiotensin 2 (AT-2) causes contractions by stimulating AT-2 receptor type 1 on vascular smooth muscle [4]. ...
Vasospasm is one of the important causes of morbidity in free flap and replantation surgery. In secondary Raynaud’s phenomenon, nearly half of the patients experience digital ulceration, pain and loss of function at least once in their lifetime. The aim of this study is to investigate the vasodilation effect of ethanol-mediated chemical denervation on peripheral vessels by topical administration. In this study, 27 Wistar albino male rats weighing 250-300 grams were used. The rats were randomly divided into three groups: saline (group S, n = 8), lidocaine (group L, n = 9) and 96% ethanol (group E, n = 9). According to group, 0.1 mL saline, 0.1 mL lidocaine and 0.1 mL ethanol were applied around the rat femoral neurovascular bundle. After the application, on the 0th day and 3th weeks, femoral artery and vein diameters were measured. After 3. weeks, histopathological samples from femoral artery, vein and nerve were evaluated. On the 0th day, the mean diameter of the femoral artery and vein was similar in group E and L and higher than group S. After three weeks, the vasodilatation effect of ethanol was increased in group E. In Group L and S, the vasodilatation effect was lost. Histopathological examination showed that ethanol significantly caused perivascular inflammation and nerve degeneration compared to other agents and did not cause endothelial damage. Vasodilatation obtained by ethanol is a rapid onset and long-lasting effect. It is also inexpensive and effective for peripheral vasodilatation.
... Elevated levels of a number of vasoconstrictors have been described, including endothelin-1 and angiotensin II. 16 Interestingly, the proposed mechanism of the protective effect of the arterial grafts on disease progression and better overall patency has been attributed to the metabolically active endothelium, producing the same vasoactive and endothelial progenitor substances that defend the native vessels from progression of atherosclerosis 17 ; in addition, incorrect handling of grafts during the manipulation may lead to vasospasm. The presence of a well-developed smooth muscle layer has also been blamed for the extreme vasoreactivity of the RA grafts, but worth of notice is that the excitation-contraction coupling in the smooth muscle cells is the main contribution to the conduit's diameter adjustment to the coronary flow and the metabolic demand of the coronary circulation. ...
... Studies in patients have confirmed the observations of organ chamber studies showing an enhanced response of RA grafts to the intra-arterial infusion of 5-HT as compared with ITA grafts [15,16]; although the reasons for this hyper-reactivity are not fully understood, both the thick muscular media and the intrinsic biologic properties of the RA probably play a role. Both a1 and a2 receptors are abundantly present in the RA, as well as histamine 1, urotensin II, angiotensin I, ET, and calcitonin receptors [17][18][19][20][21][22], while b-adrenoceptors are less represented [23]. ...
Since its reintroduction in the early 1990s the radial artery has gained a major role in coronary surgery, currently representing a valid alternative to the right internal thoracic artery as a second arterial graft. However, its peculiar morphologic and functional features have both surgical and clinical critical implications that must be taken into account. In this review we summarize the current totality of evidence on the biologic characteristics of the radial artery, such as its histopathology, vasoreactivity, and remodeling, and discuss their potential implications for use as a coronary bypass conduit.
... Radial artery produces stronger vasoconstriction than ITA to virtually all vasoconstrictors studied 12—15; however vasoconstriction is equivalent when normalised to account for the different vessel diameters in most cases. However, normalised responses to both angiotensin II and vasopressin in the radial artery are stronger and more sensitive than in the ITA, and occur irrespective of the presence of endothelium [12] [14] [15]. In addition, vasopressin-induced contraction in the radial artery is comparatively resistant to milrinone and glyceryl trinitrate, two of the most commonly used vasodilator strategies [15]. ...
Vasodilator strategies used to treat bypass grafts in the operating theatre, such as nitrates, phosphodiesterase inhibitors and calcium channel antagonists have a broad but short-lived effect against a variety of vasoconstrictor stimuli. Treatments that react irreversibly with proteins modulating vasoconstriction have the advantage that their effects can last well into the postoperative period. In addition systemic effects are avoided as the treatment is localised to the treated graft. This study investigated the use of two clinically applied drugs; fluphenazine (SKF7171A, HCl), an irreversible calmodulin antagonist and minoxidil sulphate, an irreversible potassium channel opener. Treatments were tested against receptor and non-receptor-mediated contraction in the human radial artery.
Isometric tension was measured in response to angiotensin II, KCl and vasopressin in 108 radial artery rings (taken from 31 patients undergoing coronary artery bypass grafting). Control responses were compared with rings pretreated with fluphenazine or minoxidil sulphate. Vasopressin responses were also compared in the presence of glyceryl trinitrate or the reversible Rho kinase inhibitor Y27632.
Fluphenazine pretreatment significantly suppressed vasoconstriction to all agonists tested. Maximal responses to angiotensin II, vasopressin and KCl were reduced by 42+/-19%, 35+/-8% and 48+/-15% respectively, without any measurable effect on the EC(50). Minoxidil sulphate showed no discernable effect. Vasopressin-induced contraction was also reduced by high levels of glyceryl trinitrate (220 microM; 50 microg/ml) or 10 microM Y27632.
The irreversible calmodulin antagonist fluphenazine has potential to be developed as an inhibitor of contraction in arterial graft vessels. The involvement of Rho kinase indicates that other vasoconstrictors and surgical stress can sensitize radial artery to vasopressin-induced contraction. Strategies targeting this pathway also have future potential.
... After incubation with or without cerivastatin, vascular function studies were carried out as we previously reported [19,20]. An initial tension of 80 mN and 50 mN was applied to each vascular ring of RA and LITA, respectively. ...
Statins may enhance vascular function independently of effects on cholesterol. This study investigated the ability of statins to modulate the vascular recovery of arteries used as coronary bypass grafts.
Specimens of radial artery and left internal thoracic artery were obtained during coronary artery bypass grafting. The specimens were divided into vascular rings, which were incubated in the absence or presence of cerivastatin (10(-6) mol/L) for either 2 or 24 hours. Using an organ bath technique, endothelial function was examined using acetylcholine (10(-9) to 10(-5) mol/L) after contraction by 3x10(-8) mol/L of endothelin-1.
Time-related endothelial dysfunction was shown in the control group of radial artery but not in the cerivastatin group: maximal endothelium-dependent vasodilation in the control and cerivastatin groups were 56.8% +/- 10.2% and 65.9% +/- 10.1% at 2 hours and 39.4% +/- 4.7% and 68.4% +/- 5.0% (p < 0.01, vs control) at 24 hours, respectively. On the other hand, in the left internal thoracic artery, those in the control and cerivastatin groups were 38.3% +/- 8.2% and 45.0% +/- 5.5% at 2 hours and 38.1% +/- 8.2% and 56.5% +/- 8.8% at 24 hours, respectively (NS).
In radial artery, cerivastatin significantly preserved endothelium-dependent vasodilation, which diminished with time in the control group. This could have very important implications in the clinical practice of coronary artery bypass grafting.
... The endothelial function of vascular rings was assessed as previously reported [7]. Briefly, an initial tension (80 mN for RA and 50 mN for ITA) was applied to the ring segment, which was allowed to relax and to equilibrate for 30 min at a stable level of tension. ...
Techniques aimed at improving the performance of arterial conduits will maximize the clinical benefit achievable with coronary artery bypass surgery. Controlling oxidant stress could be a strategy for preventing early graft deterioration. We tested the effect of a free radical scavenger, ascorbic acid (vitamin C), on preserving the endothelium-dependent vasodilatation function in vitro of radial artery and internal thoracic artery. We also tested its effect on the amount of reactive oxygen species (ROS) generated by each graft.
Radial artery (RA, n=25) and internal thoracic (ITA, n=19) segments were obtained from coronary artery bypass grafting patients. Each segment was divided into 3-4 mm vascular rings and incubated with or without ascorbic acid (10(-3) mol/l) for 1 h or 72 h. Using the organ bath technique, the endothelium-dependent vasodilatation function was tested in vitro by the addition of cumulative concentrations of acetylcholine (10(-9)-10(-5) mol/l) following vasocontraction by endothelin-1 (3 x 10(-8) mol/l). ROS were measured by using chemiluminescence technique at 1-h and after 72 h incubation with or without ascorbic acid.
There were no differences in the vasodilatation function between control and ascorbic acid group of both arteries in the 1-hour incubation experiment. However, in the 72 h incubation experiment, ascorbic acid preserved the endothelium-dependent vasodilatation function of RA compared with control group (35.8+/-2.2% vs. 25.9+/-2.1%; P=0.005), but not ITA (39+/-3.5% vs. 40.5+/-9.3%; P=0.438). After 72 h incubation, RA generated significantly more free radicals compared with 1 h (133.7+/-151.5 vs. 16.8+/-16.8 cps/mg x 100; P=0.01); however, AA has no statistically significant effect on decreasing the amount of free radicals generated by both arteries.
In RA, ascorbic acid is able to preserve the endothelium-dependent vasodilatation function after 72 h incubation, but not after 1 h. However, the mechanism of action of AA is not completely understood. This finding could open the door for understanding the role of oxidant stress and antioxidants in preserving the endothelial function of coronary artery bypass grafts.
The orally available chymase inhibitor BAY 1142524 is currently being developed as a first‐in‐class treatment for left‐ventricular dysfunction after myocardial infarction. Results from 3 randomized, single‐center, phase 1 studies in healthy male volunteers examining the safety, tolerability, and pharmacokinetics of BAY 1142524 are summarized. In this first‐in‐human study, single oral doses of 1‐200 mg were administered in fasted state as liquid service formulation or immediate release (IR) tablets. The relative bioavailability and the effect of a high‐fat/high‐calorie meal were investigated at the 5‐mg dose. In a multiple‐dose escalation study, doses of 5‐50 mg twice daily and 100 mg once daily were given for 5 consecutive days. BAY 1142524 was safe and well tolerated and had no effects on heart rate or blood pressure compared with placebo. BAY 1142524 was absorbed with peak concentration 1‐3 hours after administration for IR tablets; it was eliminated from plasma with a terminal half‐life of 6.84‐12.0 hours after administration of liquid service formulation or IR tablets. Plasma exposures appeared to be dose‐linear, with a negligible food effect. There was only low accumulation of BAY 1142524 after multiple dosing. BAY 1142524 exhibited a pharmacokinetic profile allowing for once‐daily dosing. The absence of blood pressure effects after administration of BAY 1142524 supports the combination of this novel anti‐remodeling drug with existing standard of care in patients with left‐ventricular dysfunction after acute myocardial infarction.
As widely validated, coronary artery bypass grafting (CABG) has outstanding consequences on long-term survival due to its outcomes, such as complete myocardial revascularization and effective patency of the utilized arterial or venous conduits. Not surprisingly, it is already established that CABG represents the gold standard procedure of revascularization for extensive coronary artery disease. Surprisingly, the mechanisms of arterial graft failure highlight the fact that the patency and biology of arterial conduits improve long-term survival after CABG. Arterial conduits are muscular arteries, therefore they are susceptible to spasm and to the competitive fl ow. Furthermore, fi brointimal proliferation as a single process is more frequent for internal thoracic artery grafts versus venous grafts. Curiously, the radial artery is more sensitive in comparison with the internal thoracic artery to many vasoconstrictor agents, including norepinephrine, 5-hydroxytryptamine, angiotensin II, endothelin, potassium chloride, phenylephrine, and vasopressin. The vasoconstriction induced by endothelin and angiotensin II on the radial artery is higher than the internal thoracic artery. The over-increased reactivity of the radial artery grafts is not established, however, the intrinsic biological fi ndings and the thick muscular media could provide a part of the explanation. Non-traumatic harvesting of arterial grafts, well-controlled dyslipidemia and avoidance of using composite grafts can be helpful in maintaining the architecture of the arterial graft and may therefore improve the shortcomings and the long-term outcomes of the patients after CABG.
We reviewed 1577 consecutive patients undergoing coronary artery bypass grafting (CABG) using endoscopic harvesting of the radial artery (RA) to define our current results.
Since 2000, we have performed endoscopic RA harvest on 1577 consecutive patients; 1476 patients had isolated CABG, and 101 patients had CABG and other procedures. The mean ± SD age was 59.4 ± 9.0 years; 80.2% were men and 40% had diabetes mellitus. All data were prospectively collected. All-cause mortality was determined using the Social Security Death Index.
There were nine in-hospital or 30-day deaths, for an operative mortality of 0.57%: mortality was 0.34% in isolated CABG and 3.85% in CABG/combined procedures. The overall estimated Kaplan-Meier survival at 1, 5, and 10 years was 99%, 95%, and 88%. In 37 patients, the RAs were not harvested or were not used for grafting because of a positive Allen test, extensive calcification or dissection, intramural hematoma, and scarring from previous arterial lines or catheterization. During postoperative follow-up, five patients (0.32%) were treated for incisional infection, and there were no ischemic hand complications. Three patients had a perioperative myocardial infarction in the RA graft distribution, and 15 patients had a coronary artery reintervention in the RA graft distribution. Two other patients had a percutaneous coronary intervention of their RAs. The overall RA patency at 10 years was 82%.
Endoscopic harvest of the RA is an excellent minimally invasive conduit harvesting technique with minimal morbidity.
Chymase is a chymotrypsin-like serine protease. It has been identified as a key angiotensin converting enzyme (ACE)-independent and endothelin converting enzyme (ECE)-independent converting enzyme that generates angiotensin II and endothelin-1 (ET-1). As an inflammatory protease, chymase participates in multiple inflammatory responses in the vasculature which drive cytokine production and adhesion molecule expression. Chymase is also involved in extracellular matrix remodeling in both vascular and non-vascular tissues. Consequently, chymase has been implicated in the pathogenesis of multiple cardiovascular, immune, and inflammatory diseases. Recent studies have shown that chymase expression and activity are increased in placental trophoblasts and in the maternal vascular endothelium in women diagnosed with preeclampsia, a hypertensive disorder in human pregnancy. The present review will address the potential roles of chymase-mediated placental and vascular dysfunction in preeclampsia. The effects of chymase on inflammatory responses associated with endothelial activation/dysfunction in preeclampsia are also discussed.
Chymase, a chymotrypsin-like protease, is a non-ACE angiotensin II (Ang II) generating enzyme. We determined if maternal chymotrypsin-like protease/chymase activity was increased in women with preeclampsia (PE).
Maternal plasma was extracted from venous blood of healthy nonpregnant women, women with normal and preeclamptic pregnancies. Chymotrypsin-like protease/chymase activity was measured by a colorimetric assay. Maternal vessel chymotrypsin-like protease/chymase expression was examined by immunohistochemistry.
Maternal plasma chymotrypsin-like protease/chymase activity was significantly higher in women with PE than in nonpregnant and normal pregnant women: 0.181 +/- 0.011 vs. 0.097 +/- 0.05 (p < 0.01) and 0.132 +/- 0.013 (p < 0.05) microkat/mL. Chymotrypsin-like protease/chymase activity was markedly reduced 24 hours postpartum in women with PE, p < 0.05. Enhanced chymotrypsin-like protease/chymase expression was observed in vascular endothelium in women with PE compared with those in normal pregnancies.
Elevated maternal chymotrypsin-like protease/chymase activity and enhanced protease immunostaining in the maternal vessel endothelium may constitute the exacerbated inflammatory state and account for the increased vascular Ang II sensitivity in PE.
The expression, localisation and function of enzymes responsible for the local formation of angiotensin II in atherosclerotic and non-atherosclerotic human coronary arteries were studied. Human epicardial coronary arteries expressed mRNA for both ACE and chymase. Immunohistochemical studies revealed that ACE was localised to the vascular endothelium, and to a lesser extent the medial smooth muscle cells, in both large and small arteries. Chymase was detected in both types of vessel but was shown to be associated with mast cells. The contractions to angiotensin I in large arteries were inhibited only by a combination of quinaprilat and soyabean trypsin inhibitor. In the intramyocardial arteries the response to angiotensin I was markedly inhibited in the presence of chymostatin. These findings demonstrate that a dual pathway for the synthesis of angiotensin II is active in diseased and non-diseased coronary arteries.
Radial artery (RA) is increasingly used as graft for coronary artery bypass grafting due to its good long-term patency. However, the mechanism of peri- and post-operative spasm is still unclear. Because of that, the aim of our study is to analyze the contractility of RA and to determine whether the presence of functional endothelium alters its contractile properties. Contractions of isolated RA rings were provoked by exogenously applied vasoconstrictors or by electrical field stimulation (EFS, 20 Hz). The order of vasoconstrictors potency based on their EC50 values was as follows: angiotensin II > phenylephrine > 5-hydroxytriptamine. Presence of endothelium increased both EC50 and maximal contraction to phenylephrine and angiotensin II, but inhibited reactivity of RA to 5-hydroxytriptamine. Spontaneous rhythmic contractions (SRC, <4 mHz) and EFS-induced contractions of RA are endothelium-independent and weaker than contractions induced by exogenously applied vasoconstrictors. Our study concludes that RA shows marked sensitivity and reactivity to angiotensin II, phenylephrine, and 5-hydroxytriptamine. Further investigations are necessary to answer why angiotensin II and phenylepehrine induce stronger contractions in the presence of endothelium. In addition, SRC as well as contractions of neurogenic origin may take part in developing vascular spasm of RA.
The radial artery (RA) is gaining popularity as a bypass conduit for coronary artery bypass grafting, and its impact on clinical practice has been extensively explored. In the present article, we provide a review of postoperative hand circulation, vascular biological characteristics of the RA graft, the efficacy of vasodilator therapies, and mid-term clinical results of use of the RA graft. Fundamental studies revealed excellent vascular biological characteristics of the RA graft as a living arterial conduit, making it almost equivalent to the internal thoracic artery (ITA) graft. Clinical studies have yielded encouraging mid-term results. Most studies reported in favor of the RA graft over the saphenous vein graft with regard to patency rate, freedom from cardiac events, and survival. However, superiority of either the RA or right ITA graft has not been conclusively determined. The long-term results of RA grafts remain unknown, but at present, supplementary use of an RA graft with a left ITA graft appears feasible for CABG.
The antioxidant properties of blood vessels contribute to their performance and patency of that vessel when used as a bypass conduit. Despite increased use of the radial artery (RA) in recent years, very little is known about its antioxidant properties. We compared the ability of the RA to generate superoxide and assessed its antioxidant protective capacity with that of the internal thoracic artery (ITA).
Vascular segments of the ITA and the RA were obtained from patients undergoing coronary artery bypass grafting (CABG) incubated in culture media for 2, 24, 48 and 72 hours. The amount of superoxide generated by each artery, and the deterioration of the endothelial function were assessed by using chemiluminescence (CL) and organ bath techniques. We also assessed the expression, localisation and the activity of superoxide dismutase (SOD) in both arteries; using reverse transcription-polymerase chain reaction (RT-PCR), immunolocalisation techniques and standard biochemical assessment of SOD activity.
Under stress, the RA generated more superoxide (133.6+/-54.7 at 72h vs. 16.8+/-6.4 at 2h; P<0.01) and its endothelial function deteriorated faster (56.3+/-7.3 at 72h vs. 20.2+/-1.5 at 2h; P<0.0001) than that of ITA. Cu/Zn-SOD was found to be prevalent in the endothelium, while Ec-SOD was distributed evenly in the endothelium and media of both arteries. The activity of SOD was less in the RA compared with that of the ITA (510.2+/-219.8 vs. 808.6+/-343.7, respectively; P=0.03).
Our study shows that the RA is less equipped with an antioxidant protective mechanism compared with the ITA. These findings could partially explain the differential clinical performance of these conduits in CABG.
Diadenosine polyphosphates (Ap(n)A) are released by degranulating platelets and high, local concentrations may form at sites of platelet activation. Radial artery grafts, now often used alongside the internal mammary artery in coronary artery bypass surgery, are particularly reactive to several vasoconstrictors but the response to Ap(n)A has not been investigated. This study compared the vasoconstrictor activity of Ap(n)A in human radial artery with other vessels commonly used as bypass grafts. Radial artery demonstrated robust concentration-dependent vasoconstriction to Ap(n)A (n=4-6) at concentrations in the micromolar range. In contrast, average responses in internal mammary artery were negligible. Cross-desensitization revealed that Ap(n)A-mediated vasoconstriction occurred via an alphabetamethyleneATP-sensitive receptor. Responses to both Ap(5)A and alphabetamethyleneATP were inhibited by suramin but were insensitive to the P2X(1) receptor antagonist 8,8'-[Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid (NF279). Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) enhanced responses to Ap(5)A. Similar responses were obtained in saphenous vein. In conclusion, diadenosine polyphosphates contract radial artery and saphenous vein by an as yet uncharacterized P2X receptor but have only limited activity in internal mammary artery. The selective activity of diadenosine polyphosphates in radial artery would implicate them as potential mediators of post-operative contraction in this graft.
The radial artery has increased in popularity as a conduit for use in coronary artery bypass surgery. However, concerns remain regarding the risk of radial artery spasm. Although the use of different pharmacological agents to prevent and treat this has been described, there is currently no clear agreement as to the optimal agent. To clarify which agents are most suitable for clinical use, all pertinent studies to date (January 2007) that have reported the efficacy of pharmacological agents in the prevention and treatment of radial artery spasm have been reviewed. It can be argued that verapamil-glycerine tri-nitrate solution represents the optimum agent when used in the perioperative period.
Overlapping genomic clones containing the entire sequence of the human angiotensin I-converting enzyme (ACE) gene were isolated from a lamda phage human DNA library. This gene spans 21 kilobases (kb) and comprises 26 exons, ranging in size from 88 to 481 base pairs. Intron-exon boundaries were sequenced and the relative positions of the exons were mapped. The two different mRNAs transcribed from the ACE gene were assigned to their respective exons. The large endothelial type ACE mRNA (4.3 kb long) is transcribed from exon 1 to exon 26, excluding exon 13. The 3-kb long testicular ACE mRNA is transcribed from exon 13 to exon 26. Exon 13 encodes for the 67 amino acids of the NH2-terminal region of the testicular ACE, whereas downstream exons encode a sequence common to both isozymes. The gene duplication suggested by the internal homology of the endothelial ACE mRNA is now confirmed by the presence of two homologous clusters of eight exons (exons 4-11 and exons 17-24) having similar sizes and codon phases at exon-intron boundaries. The presence of two alternate promoters was investigated by ribonuclease protection assays. The different 5' ends of the two ACE transcripts revealed a promoter for the endothelial ACE mRNA in the 5'-flanking region of the first exon and a promoter for the testicular ACE mRNA situated in intron 12.
We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.
Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.
We report the complete nucleotide sequence of a human beta actin cDNA. Both the 5' and 3' untranslated regions of the sequence are similar (greater than 80%) to the analogous regions of the rat beta-actin gene reported by Nudel et al (1983). When a segment of the 3' untranslated region is used as a radiolabelled probe, strong hybridization to chick beta actin mRNA is seen. This conservation of sequences suggests that strong selective pressures operate on non-translated segments of beta actin mRNA.
Using isolated arteries, we demonstrated a marked difference in the angiotensin II-forming systems between human and rodent vessels. In human arteries, only 30-40% of the conversion of angiotensin I to angiotensin II depended on the angiotensin-converting enzyme (ACE), and the rest of the angiotensin II formation was ascribed to chymostatin-sensitive angiotensin II-generating enzyme (CAGE). On the contrary, angiotensin II formation in rodent arteries totally depended upon ACE, without any sign of CAGE involvement. Such a marked species-difference can be relevant to the reported difference between humans and rodents in the ACE inhibitor effects on the myointimal hyperplasia after intimal balloon injury.
Arterial grafting for the correction of coronary artery disease preceded the use of saphenous vein grafts, but the overwhelming popularity of the saphenous vein from 1970 to 1985 left the development of arterial grafting dormant. Excellent graft patency results from pedicled internal thoracic artery grafting and continued saphenous vein graft failure prompted our unit to explore complete arterial grafting with internal thoracic artery and radial artery grafts. One thousand and fifty-three patients who received a combination of internal thoracic artery and radial artery grafts were compared with 1,156 patients who received internal thoracic artery and saphenous vein grafts. All patients underwent primary coronary artery bypass surgery between 1995 and 1998. The early mortality and morbidity and the probability of survival at 2 years were similar in both groups of patients. Early graft patency studies of 35 radial artery grafts showed 33 (94%) were patent at a mean of 12 months. Complete arterial grafting using internal thoracic and radial arteries is safe and may provide a long-term benefit.
Of 22 patients undergoing repeat open-heart surgery through a previous median sternotomy wound 11 were randomised to receive the serine proteinase inhibitor aprotinin in high dosage (about 700 mg intravenously from the start of anaesthesia to the end of operation, depending on the length of the surgical procedure). Their mean blood loss was 286 ml compared with 1509 ml in the 11 control patients (p less than 0.001), and mean haemoglobin losses were 8.3 g and 78 g, respectively (p less than 0.001). Blood transfusion requirements were eightfold higher in the control group than in the aprotinin group, 7 of whom received only the single unit of their own blood taken before cardiopulmonary bypass.
Dog, monkey and human aortic tissues contained two distinct types of angiotensin II-generating enzymes; angiotensin converting enzyme (ACE) and chymostatin-sensitive angiotensin II-generating enzyme (CAGE). Endothelium, media and adventitia of canine thoracic aortae were separated using collagenase digestion, and determined for their ACE and CAGE activity. ACE activity was assayed by hippuryl-His-Leu cleavage. CAGE activity was estimated with ANG I as substrate in the presence of inhibitors of ACE and angiotensinases. His-Leu, the common product of both enzyme reactions, was fluorimetrically quantified after o-phthalaldehyde condensation. ACE localized mainly in endothelium, while CAGE distributed predominantly in adventitia. Similar results were obtained with human and monkey aortae. Such a contrasting distribution may indicate the distinct functional role of these two enzymes.
Human chymase is a potent and specific angiotensin (Ang) II-forming serine proteinase. Although the histological localization of heart chymase indicated that this enzyme contributes to extracellular Ang II formation, the systemic distribution and the level of expression of chymase in various human tissues have not been clarified. This information is needed to elucidate the human tissue Ang II system.
Levels of immunoreactivity and enzymatic activity in various human tissues were evaluated respectively by Western blot analysis and by an enzymatic assay for Ang II-forming activity from Ang I.
High levels of chymase-like immunoreactivity were found in alimentary tract tissue, uterus and tonsil; moderate levels were found in both cardiac ventricles, lung, adenoid and liver; low levels were found in the cardiac atria, coronary artery, aorta and skin; and almost undetectable levels were found in the spleen and kidney. High levels of chymase-like enzymatic activity were detected in skin, oesophagus, stomach and uterus; moderate levels were found in both cardiac ventricles, lung, colon, tonsil, adenoid and renal cortex; and low levels were found in the cardiac atria, coronary artery, aorta, spleen, renal medulla and liver.
Our studies have revealed heterogeneous and widespread tissue distribution of human chymase throughout the human body and indicate that chymase probably has a significant influence not only in the heart but also in other tissues.
GROWTH-PROMOTING EFFECTS OF ANGIOTENSIN: Angiotensin, a vasoconstrictive peptide, is now known to be an agent of vascular and cardiac growth and may directly influence the pathophysiology of coronary artery disease and ventricular remodeling. Vascular growth occurs when angiotensin activates autocrine and paracrine growth factors, including fibroblast growth factor, transforming growth factor beta-1 and platelet-derived growth factor, and is modulated by endothelium-derived vasodilators and growth inhibitors. ANGIOTENSIN AND CARDIOVASCULAR DISEASE: The presence of angiotensin converting enzyme (ACE) and angiotensin II has been demonstrated in vascular tissue, and these local substances are causally involved in the development of vascular lesions. Similarly, angiotensin can stimulate cardiac myocyte growth and matrix modulation. Cardiac tissue ACE is implicated in ventricular remodeling in the course of progressive heart failure. A genetic variant of the ACE gene has been reported to be associated with increased risks of cardiovascular pathology. ACE INHIBITOR THERAPY: To date, studies of ACE inhibitor treatment in human patients have not demonstrated any prevention of restenosis after angioplasty. However, recent clinical trials in postmyocardial infarction reported that ACE inhibitor therapy reduces recurrent myocardial infarction and prevents cardiac enlargement. Long-term prospective trials are currently being conducted to examine the effects of ACE inhibitor therapy on coronary ischemic events and coronary atherosclerosis, as evaluated by angiography or intravascular ultrasound, and the relationship between coronary events and ACE gene polymorphism.
Recently, satisfactory results were obtained in a series of patients in whom the radial artery was used as a conduit for coronary artery bypass. However, spasm of this conduit was observed in 4% of patients. The aim of this study was to analyze the vasoreactive properties of the radial artery and to compare them with those of the internal mammary and the gastroepiploic arteries.
Human radial (56 from 15 patients), internal mammary (77 from 20 patients), and gastroepiploic (41 from 12 patients) artery ring segments were mounted on a strain gauge in oxygenated, normothermic Krebs' solution at optimal resting tension. With KCl (100 mM) serving as the control, the dose-response curves to norepinephrine, serotonin, and thromboxane A2 mimetic were obtained, permitting assessment of force of contraction and sensitivity. Functional endothelium was assessed with acetylcholine. Smooth muscle-dependent relaxation was assessed with sodium nitroprusside. The radial artery had stronger contractions to KCl than the other vessels. The radial and the gastroepiploic arteries with endothelium presented a higher contraction force than the internal mammary artery in response to norepinephrine and serotonin. The three vessels had equal sensitivities to norepinephrine and serotonin. The gastroepiploic artery had a lower sensitivity to thromboxane A2 mimetic than the two other vessels.
This increased reactivity of the radial artery explains its propensity to spasm and emphasizes the need for antispastic drugs and platelet inhibitors when the radial artery is used for coronary artery bypass.
Previously, we had reported that 7-day administration of the angiotensin-converting enzyme inhibitor quinapril markedly reduced electrically evoked pressor responses in the isolated, perfused mesenteric vascular bed of the spontaneously hypertensive rat (SHR). In the present study, we investigated the possibility that quinapril alters postsynaptic vasoconstrictor activity to a variety of vasoconstrictive agents. Quinapril (10 mg/kg/day), administered orally to SHR for 7 days, significantly reduced the potency and the maximal vasopressor response to phenylephrine (2.5-fold and 40%, respectively) and the maximal response to serotonin (50%) compared with the responses from vehicle treated SHR. In contrast, quinapril had no significant effect on the pressor responses to KCl or phorbol ester. Furthermore, an equipotent antihypertensive dose of hydralazine (5 mg/kg/day, p.o., for 7 days) exerted no inhibitory effect on the pressor responses elicited by phenylephrine, KCl phorbol ester, but significantly reduced the maximal response to serotonin. In addition, vasopressor responses to phenylephrine were not affected by an acute (i.e., 75 min) infusion of quinaprilat, the active metabolite of quinapril. The results suggest that 7-day quinapril administration, and not acute treatment, reduces alpha 1 adrenoceptor and S2-serotonergic receptor-mediated vasoconstriction. However, quinapril did not reduce the vasoconstrictor responses induced by KCl or phorbol ester, indicating that those pressor responses that are due to depolarization or protein kinase C activation are, in part, independent of angiotensin-converting enzyme inhibition. This inhibition of vascular alpha 1 adrenoceptor and S2-serotonergic receptor pressor activity may underlie, in part, the long-term antihypertensive activity of quinapril in the SHR.
Background:
The radial artery (RA) has been used as an alternative arterial graft for coronary artery bypass grafting. This artery has been reported to be spastic; therefore, use of spasmolytic drugs (vasodilators) during the preparation of the RA is recommended. The combination of calcium antagonists and nitroglycerin (verapamil plus nitroglycerin, VG solution) has been suggested to be effective in other bypass grafts. This study was designed to investigate (1) the effect of the VG solution during preparation of the RA for coronary artery bypass grafting and (2) whether the effect would last for a prolonged period after topical use.
Methods:
Ring segments of the RA taken from coronary artery bypass grafting patients were studied in organ chambers at a physiologic pressure. The relaxation effect of VG (30 mumol/L verapamil and 30 mumol/L nitroglycerin, n = 5) or papaverine (30 mumol/L, n = 5) was recorded in K+ (25 mmol/L potassium chloride)-precontracted RA segments. Other segments taken from the RA were treated (n = 5) or not treated (n = 20, as the control) with the VG solution for 45 minutes before used for coronary artery bypass grafting. Both the VG-treated and nontreated segments were immersed in oxygenated Krebs solution and stored in a refrigerator at 4 degrees C for 24 hours. The contraction force to 100 mmol/L K+ was compared between the two groups.
Results:
The VG solution induced more rapid relaxation than papaverine (p < 0.05 from the first to the ninth minute). After 10 minutes, both solutions induced full relaxation. The contraction to K+ (100 mmol/L) in rings treated with VG solution for 45 minutes during operation was almost abolished 24 hours later (0.86 +/- 0.1 g). In contrast, the control segments (n = 20) taken from nontreated RA contracted with K+ to high force (5.0 +/- 0.6 g; p < 0.001).
Conclusions:
The present study suggests that the combination of verapamil and nitroglycerin may provide a rapid onset, a complete relaxation, and a long-lasting vasorelaxant effect when used to prepare the RA for grafting. This study adds a new method to prevent RA spasm during coronary artery bypass grafting.
Angiotensin may play a pathophysiological role in experimental and human cardiovascular disease. Clinical studies have shown that ACE inhibitors reduce mortality, recurrent myocardial infarction, and ischemic events in patients with left ventricular dysfunction. Animal studies suggest that tissue ACE, particularly within blood vessels, may be an important target.
To study tissue ACE in human coronary artery disease and to identify potential mechanisms of ACE inhibitor action, we examined ACE expression immunohistochemically in nonatherosclerotic and diseased human coronary arteries. In nonatherosclerotic arteries, ACE immunoreactivity was found in luminal and adventitial vasa vasorum endothelium. In early- and intermediate-stage atherosclerotic lesions, ACE was detected prominently in regions of fat-laden macrophages and in association with T lymphocytes. In advanced lesions, ACE immunoreactivity was also localized to the endothelium of the microvasculature throughout the plaques. Immunoreactive angiotensin II was also detected in these areas. ACE expression in macrophages was further examined by in vitro experiments with a monocytoid cell line. ACE activity was induced threefold after differentiation of the cells into macrophages and was further increased after stimulation with acetylated LDL.
These observations demonstrate that significant sources of tissue ACE in human atherosclerotic plaques are regions of inflammatory cells, especially areas of clustered macrophages as well as microvessel endothelial cells. These results suggest that ACE accumulation within the plaque may contribute to an increased production of local angiotensin that may participate in the pathobiology of coronary artery disease. Plaque ACE probably is an important target of drug action.
Blockade of the renin-angiotensin system by inhibition of angiotensin-converting enzyme (ACE) is beneficial for the treatment of hypertension and congestive heart failure. However, it is unclear how complete the blockade by ACE inhibitors is and if there is continuing angiotensin II (Ang II) formation during chronic treatment with ACE inhibitors. Indeed chymase, a serine protease, which is able to form angiotensin II from angiotensin I (Ang I) and cannot be blocked by ACE inhibitors, has been shown to be present in human heart. The goal of the present study was to evaluate the extent of renin-angiotensin system blockade and the Ang II-forming pathways in cardiac tissue of patients chronically treated with ACE inhibitors or in patients without ACE inhibition therapy. Our studies indicate an incomplete ACE inhibition in human heart tissue after chronic ACE inhibitor therapy. Moreover, ACE contributes only a small portion to the total Ang I conversion, as shown in biochemical studies in ventricular and coronary homogenates or functionally as Ang I contractions in isolated rings of coronary arteries. A serine protease was responsible for the majority of Ang II production in both the membrane preparation and Ang I-induced contractions of isolated coronary arteries. In humans, the serine protease pathway is likely to play an important role in cardiac Ang II formation. Thus, drugs such as renin inhibitors and Ang II receptor blockers might be able to induce a more complete blockade of the renin-angiotensin system, providing a more efficacious therapy.
Data from in vitro studies suggest that both chymase and ACE contribute to the local generation of angiotensin (Ang) II in the heart. The enzyme kinetics under in vivo conditions are unclear. We thus studied the generation of Ang II by cardiac tissue in the presence of interstitial fluid (IF) that contains a variety of naturally occurring protease inhibitors.
Ang I was incubated with heart homogenate in the presence of IF. IF obtained from human skin contained substantial amounts of protease inhibitors and ACE activity, the concentration of alpha 1-antitrypsin being 35% and the activity of ACE 24% of the corresponding serum values. When heart homogenate was incubated with Ang I, three enzymes were responsible for its metabolism: heart chymase and heart ACE converted Ang I to Ang II, and heart carboxypeptidase A (CPA)-like activity degraded Ang I to Ang-(1-9). Incubation of heart homogenate in the presence of IF led to practically full inhibition of heart chymase-mediated Ang II formation by the natural protease inhibitors present in IF. In contrast, heart CPA-like activity was not blocked, as reflected by the continued generation of Ang-(1-9). In addition, both heart ACE- and IF ACE-mediated Ang II formation were strongly inhibited. This inhibition was shown to be due to the Ang-(1-9) formed.
The present experimental study defines two novel inhibitory mechanisms of Ang II formation in the human heart interstitium. Heart chymase-mediated Ang II formation is strongly inhibited by the natural protease inhibitors present in the IF. Similarly, both heart ACE- and IF ACE-mediated Ang II formation appear to be inhibited by the endogenous inhibitor Ang-(1-9) formed by heart CPA-like activity. These inhibitory mechanisms provide additional information about how the Ang II concentration in the heart interstitium may be controlled.
The radial artery (RA) has been used as an alternative arterial graft for coronary artery bypass grafting, but this artery has been suggested to be spastic. Endothelin-1 (ET) and angiotensin II (AII) have been measured with increased plasma concentrations during cardiopulmonary bypass. However, whether RA is reactive to these two important receptor-mediated vasoconstrictors is unknown. Also unknown is the endothelial function of this arterial conduit. This study was designed to compare RA and the internal mammary artery regarding the contractile characteristics to ET-1 and AII and endothelial function.
Ring segments of the RA and internal mammary artery taken from patients undergoing coronary artery bypass grafting were studied in organ chambers at a physiologic pressure. The contractility was determined from the contraction induced by ET-1 and AII as contraction force and the force normalized by circumference (g/mm). The endothelium-dependent relaxation was induced by the calcium ionophore A23187, a nonreceptor agonist, and substance P, a receptor agonist for endothelium-derived relaxing factors. Nitroglycerin was used to study the endothelium-independent relaxation.
Both ET-1 and AII induced a higher contraction force (9.0 +/- 0.9 g, n = 12, versus 4.5 +/- 0.4 g, n = 38, p < 0.0001 for ET and 6.5 +/- 1.9 g, n = 7, versus 1.7 +/- 0.3 g, n = 8, p = 0.015 for AII) and normalized force (0.95 +/- 0.1 g/mm versus 0.66 +/- 0.05 g/mm, p = 0.007 for ET-1 and 0.8 +/- 0.2 g/mm versus 0.2 +/- 0.05 g/mm, p < 0.01 for AII) in RA than in the internal mammary artery. There were no significant differences detected between these arteries with regard to either endothelium-dependent (to substance P and A23187) or endothelium-independent (to nitroglycerin) relaxation (p > 0.05).
We conclude that the human RA has a higher receptor-mediated contractility (to ET-1 and AII) but similar endothelial function compared to the internal mammary artery. The study reveals the nature of the more spastic characteristics of the RA, supports the necessity of a more active pharmacologic intervention to relieve spasm in the RA, and suggests that the similar endothelium-derived relaxing factor-mediated endothelial function of the RA compared with the internal mammary artery may be the basis for a satisfactory long-term patency.
Internal mammary artery (IMA) bypass grafts have a higher patency than saphenous vein (SV) grafts. Intimal hyperplasia of SV grafts is due to smooth muscle cell (SMC) proliferation and migration. We hypothesized that different SMC growth activity exists in IMA and SV, which may explain the different patencies of arterial and venous grafts.
SMCs were isolated from IMA and SV by explant culture and stimulated with serum or platelet-derived growth factor-BB (PDGF-BB). Cell growth was analyzed by explant outgrowth rate, 3H-thymidine incorporation, or cell counting. PDGF receptor expression and autophosphorylation, regulation of mitogen-activated protein kinases (MAPKs), and cyclin-dependent kinase inhibitors (p27Kip1 and p21Cip1) were analyzed by molecular techniques. SMC outgrowth from explants by serum (20%) over a 20-day period was more pronounced in SV (37+/-5%) than in IMA (4+/-3%; P<.001) of the same patients. Serum (10%) increased cell number more rapidly in SV (2 x 10(4)/well to 18+/-4 x 10(4)/well; P<.05) than in IMA (2 x 10(4)/well to 9+/-4 x 10(4)/well; P<.05) over an 8-day period. PDGF-BB (0.01 to 10 ng/mL) stimulated 3H-thymidine incorporation (1347+/-470% above control levels) and increased cell number in SV (2 x 10(4)/well to 5+/-1 x 10(4)/well; P<.05) but not in IMA. PDGF alpha- and beta-receptors were similarly expressed and were activated in both SV and IMA. PDGF-BB induced a similar MAPK activation (kinetics and maximal activity) in both SV and IMA cells but increased MAPK protein level only in SV. Furthermore, PDGF-BB markedly downregulated the cell cycle inhibitor p27Kip1 in SV, but this was much less pronounced in IMA.
SMCs from SVs exhibit enhanced proliferation compared with IMA in spite of functional growth factor receptor expression and MAPK activation. However, PDGF increased MAPK protein level only in SV and downregulated cell cycle inhibitor (p27Kip1) more potently in SV than in IMA. This may explain the resistance to growth stimuli of IMA SMCs and may contribute to the longer patency of arterial versus venous grafts.
The activity of the renin-angiotensin system may be important in determining the performance of coronary artery bypass grafts. We have examined the activity of tissue angiotensin-converting enzyme and the effects of angiotensin II in vessels used as bypass grafts.
Organ bath studies were used to determine the vasoactive effect of angiotensin II. The activity of the angiotensin-converting enzyme was assessed by metabolism of a specific synthetic substrate.
The saphenous vein produced greater maximum responses to angiotensin II than did the internal thoracic artery. This response was not modified by inhibition of nitric oxide synthase, cyclooxygenase, or by an endothelin receptor antagonist in either vessel. Losartan, an AT1 receptor antagonist, inhibited the vasoconstrictor response in both blood vessels. Homogenates of saphenous vein and internal thoracic artery displayed tissue angiotensin-converting enzyme activity, which was inhibited by captopril. Enzyme activity was threefold greater in the vein. Both the contractile response to angiotensin II and the enzyme activity were retained in venous grafts removed up to 20 years after coronary bypass surgery.
These data demonstrate that marked differences exist in angiotensin-converting enzyme activity and AT1 receptor responses in the saphenous vein compared with the internal thoracic artery. These findings may have important implications for the performance of the vein when used as a coronary artery bypass graft and may have clinical implications for the use of angiotensin-converting inhibitors and AT1 receptor antagonists in the prevention and treatment of vein graft disease.
The aim of our study was to demonstrate the existence, location and functional importance of an alternative angiotensin II‐forming pathway other than angiotensin converting enzyme (ACE) in the human saphenous vein (SV).
Vascular reactivity studies using an in vitro organ bath technique showed that the SV ( n =20) produced similar maximum contractions in response to angiotensin I (41.5±5.4 mN) compared to those observed to angiotensin II (46.7±10.9 mN). The response to angiotensin I could be significantly inhibited ( P <0.05 ) by incubation with the AT 1 receptor antagonist losartan (1 μ M ).
Prior incubation of segments of SV with either captopril (1 μ M ) ( n =6), quinaprilat (1 μ M ) ( n =7), or the chymase inhibitor soya bean trypsin inhibitor (SBTI) (10 μ M ) ( n =7) singularly failed to have any inhibitory effect on the response to angiotensin I. However when vessel segments ( n =7) were co‐incubated with quinaprilat (1 μ M ) and SBTI (10 μ M ), the SV exhibited a rightward shift in curve profile to angiotensin I and a markedly reduced maximum response 12.5±2.4 mN, when compared to control (30.4±7.6 mN), quinaprilat (24.5±9.4 mN), and SBTI (31.6±10.7 mN) on their own.
An immunohistochemical technique employing streptavidin biotin peroxidase localised ACE to both endothelial cells and smooth muscle cells while chymase was confined to mast cells in the adventitia of the vessel wall.
In conclusion, our results demonstrate the existence of an alternative angiotensin I converting pathway to that of ACE, involving chymase. Therefore, there is the capacity for a continuation of angiotensin II formation. in the presence of ACE inhibition.
British Journal of Pharmacology (1998) 125 , 423–428; doi: 10.1038/sj.bjp.0702018
Angiotensin converting enzyme (ACE) is thought to be the main enzyme to convert antiotensin I to the vasoactive angiotensin II. Recently, in the human heart, it was found that the majority of angiotensin II formation was due to another enzyme, identified as human heart chymase. In the human vasculature however, the predominance of either ACE or non‐ACE conversion of angiotensin I remains unclear.
To study the effects of ACE‐ and chymase‐inhibition on angiotensin II formation in human arteries, segments of internal mammary arteries were obtained from 37 patients who underwent coronary bypass surgery.
Organ bath experiments showed that 100 μ M captopril inhibited slightly the response to angiotensin I (pD 2 from 7.09±0.11–6.79±0.10, P <0.001), while 100 μ M captopril nearly abolished the response to [pro ¹⁰ ] angiotensin I, a selective substrate for ACE, and the maximum contraction was reduced from 83±19%–23±17% of the control response ( P =0.01). A significant decrease of the pD 2 of angiotensin I similar to captopril was observed in the presence of 50 μ M chymostatin (pD 2 from 7.36±0.13–6.99±0.15, P <0.039), without influencing the maximum response. In the presence of both inhibitors, effects were much more pronounced than either inhibitor alone, and a 300 times higher dose was needed to yield a significant contraction response to angiotensin I.
These results indicate the presence of an ACE and a non‐ACE angiontensin II forming pathway in human internal mammary arteries.
British Journal of Pharmacology (1998) 125 , 1028–1032; doi: 10.1038/sj.bjp.0702150
In vitro studies of myocardial tissue suggest that angiotensin II (Ang II) may be generated by both ACE and chymase. A similar dual pathway may exist in the vasculature. We studied the effects of ACE and chymase inhibitors on the contractile response to angiotensin I (Ang I) in human resistance arteries to investigate ACE-independent generation of Ang II.
Subcutaneous resistance arteries (250 to 350 microm) were obtained from gluteal biopsies from volunteers and New Zealand White rabbits and mounted on a wire myograph. Contractile ability was tested with high-potassium depolarization and norepinephrine 10 micromol/L and endothelial integrity by relaxation to acetylcholine 3 micromol/L. Cumulative concentration-response curves were constructed for Ang I in the presence of enalaprilat 1 micromol/L, chymostatin 10 micromol/L, or both inhibitors together. In the rabbit, enalaprilat completely inhibited the Ang I response. In human vessels, enalaprilat or chymostatin alone had no effect, but the combination of enalaprilat and chymostatin almost completely inhibited the response to Ang I.
A dual pathway for Ang II generation exists in human resistance arteries, mediated by ACE and a chymostatin-sensitive enzyme, probably chymase. We confirm that a marked species difference exists in the mechanism of Ang II generation between the human and the rabbit. More efficacious suppression of the renin-angiotensin system may require development of novel enzyme inhibitors or combinations of currently available drugs.
Angiotensin II may contribute to atherogenesis by facilitating the proliferative and inflammatory response to hypercholesterolemia. This study determined, in a primate model of diet-induced atherosclerosis, the effect of AT(1) blockade on fatty-streak formation, plasma lipids, and surrogate markers of vascular injury.
Male cynomolgus monkeys fed a diet containing 0.067 mg cholesterol/kJ for 20 weeks were given losartan (180 mg/d, n=6) or vehicle (n=8) for 6 weeks starting at week 12 of the dietary regimen. Arterial pressure, heart rate, plasma total and lipoprotein cholesterol concentrations, and lipoprotein particle sizes and subclass distributions were unaffected by treatment. Losartan caused significant (P<0.05) increases in plasma angiotensin II and angiotensin-(1-7). Compared with vehicle-treated controls, losartan reduced the extent of fatty streak in the aorta, the coronary arteries, and the carotid arteries by approximately 50% (P<0.05). A significant (P<0.05) reduction in the susceptibility of LDL to in vitro oxidation, serum levels of monocyte chemoattractant protein-1, and circulating monocyte CD11b expression were also associated with losartan treatment. In addition, serum levels of vascular cell adhesion molecule-1 and E-selectin did not change during treatment but increased after discontinuation of losartan. Serum C-reactive protein, platelet aggregability, and white cell counts were not modified by losartan.
This study demonstrates for the first time an antiatherogenic effect of AT(1) receptor blockade in nonhuman primates. Losartan inhibited fatty-streak formation through mechanisms that may include protection of LDL from oxidation and suppression of vascular monocyte activation and recruitment factors.
Structural and functional alterations of the vasculature may contribute to complications of hypertension. Because angiotensin II may be pivotal in some of these vascular abnormalities, we tested the hypothesis that the angiotensin type 1 (AT(1)) receptor antagonist losartan, in contrast to the beta-blocker atenolol, would correct resistance artery abnormalities in patients with essential hypertension.
Nineteen untreated patients with mild essential hypertension (47+/-2 years, range 30 to 65 years; 57% male) were randomly assigned in double-blind fashion to losartan or atenolol treatment for 1 year. Nine age/sex-matched normotensive subjects were also studied. Both treatments reduced blood pressure to a comparable degree (losartan, from 149+/-4.1/101+/-1.6 to 128+/-3.6/86+/-2.2 mm Hg, P<0.01; atenolol, from 150+/-4.0/99+/-1.2 to 130+/-3.2/84+/-1.4 mm Hg, P<0.01). Resistance arteries (luminal diameter 150 to 350 microm) dissected from gluteal subcutaneous biopsies were studied on a pressurized myograph. After 1 year of treatment, the ratio of the media width to lumen diameter of arteries from losartan-treated patients was significantly reduced (from 8.4+/-0.4% to 6.7+/-0.3%, P<0.01). Arteries from atenolol-treated patients exhibited no significant change (from 8. 3+/-0.3% to 8.8+/-0.5% after treatment). Endothelium-dependent relaxation (acetylcholine-induced) was normalized by losartan (from 82.1+/-4.9% to 94.7+/-1.1%, P<0.01) but not by atenolol (from 80. 4+/-2.7% to 81.7+/-4.6%). Endothelium-independent relaxation (by sodium nitroprusside) was unchanged after treatment.
The AT(1) antagonist losartan corrected the altered structure and endothelial dysfunction of resistance arteries from patients with essential hypertension, whereas the beta-blocker atenolol had no effect.
Activity of the renin angiotensin system in human bypass grafts
- Crabbe
Evolutionary conservation in the untranslated regions of actin mRNAs.
- Ponte P.
- Ng S.Y.
- Engel J.
- Gunning P.
- Kedes L.
Evolutionary conservation in the untranslated regions of actin mRNAs
- Ponte