Impact of type 2 diabetes on vascular reactivity to cGMP generators in human internal thoracic arteries.

OBJECTIVE: The balance between nitric oxide (NO)-sensitive and -insensitive forms of soluble guanylate cyclase (sGC) has been demonstrated to be disrupted in certain lifestyle-related diseases. However, it remains unclear whether type 2 diabetes results in a shift of sGC to the NO-insensitive form. This study addressed this issue in the human blood vessel. METHODS: Internal thoracic arteries were obtained from patients undergoing coronary artery bypass grafting. Helically cut strips of the arteries were suspended in organ chambers, and relaxant responses to nitroglycerin (NO-sensitive sGC stimulant) and BAY 60-2770 (NO-insensitive sGC stimulant) were assessed. RESULTS: The patients were divided into two groups according to the presence of type 2 diabetes (HbA1c: 7.0±0.3%) or its absence (HbA1c: 5.6±0.1%). Nitroglycerin-induced relaxation was not different in the arteries obtained from type 2 diabetic and non-diabetic patients. In addition, the relaxant response to BAY 60-2770 in type 2 diabetics was comparable to that observed in non-diabetics. Although the patients enrolled often had vascular risk factors other than type 2 diabetes, the relaxant responses were still in the same range in a comparison based on the number of risk factors. However, in separate experiments, the relaxant response to nitroglycerin was attenuated by pre-incubation of the arteries with ODQ (sGC imbalance inducer), whereas the relaxant response to BAY-60-2770 was augmented. CONCLUSIONS: These findings suggest that type 2 diabetes does not affect the balance between NO-sensitive and -insensitive sGC in human internal thoracic artery grafts.

In vitro effects of sodium nitroprusside and leptin on norepinephrine-induced vasoconstriction in human internal mammary artery.

AIM: The biological and pharmacological properties of vessels used in coronary artery bypass graft (CABG) surgery are as important as their mechanical properties. The aim of this study was to investigate the possible role of protein kinase C (PKC)-dependent mechanisms in leptin-induced relaxation in the human internal mammary artery (IMA). METHODS: IMA rings, obtained from patients undergoing CABG surgery, were suspended in isolated tissue baths containing Krebs-Henseleit solution, which were continuously gassed with 95% O2 and 5% CO2 at 37(°)C. RESULTS: The IMA rings were pre-contracted with increasing concentrations of norepinephrine (NE 10(-9)-10(-4) mol/l) and the relaxation responses to sodium nitroprusside (SNP), a nitrosovasodilator, and leptin were studied in the presence and absence of a PKC inhibitor. Leptin (1 µM) caused a dose-dependent relaxation in NE pre-contracted IMA rings. Pre-treatment with a PKC inhibitor significantly attenuated this vasorelaxatory response to leptin in human isolated IMA. CONCLUSION: It was found that SNP and leptin caused significant relaxation of the NE pre-contracted human IMA rings, and PKC was probably the sub-cellular mediator for this effect. Our findings may have clinical or pharmacological importance as it could be hypothesised that obese subjects who have a left IMA bypass graft would have better myocardial perfusion.

Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: possible role of differential RhoA activity.

BACKGROUND/PURPOSE: Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. METHODS/MATERIALS: SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. RESULTS: IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P<0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P<0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P<0.05 and 43% reduction in activity, P<0.01). CONCLUSIONS: IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients. SUMMARY: The internal mammary artery (IMA) is the conduit of choice for bypass grafting and is generally successful in all patients, including those with type 2 diabetes (T2DM). By contrast, saphenous vein (SV) is inferior to IMA and furthermore patients with T2DM suffer strikingly poorer outcomes than their non-diabetic (ND) counterparts. We discovered that SV-SMC from T2DM patients exhibit altered persistent morphology and function compared to ND SV-SMC, with differential expression and activity of the small GTPase RhoA, yet ND and T2DM IMA-SMC were indistinguishable. These data offer an explanation for the superior patency of IMA grafting independent of the presence of diabetes.

The vasa vasorum and associated endothelial nitric oxide synthase is more important for saphenous vein than arterial bypass grafts.

No-touch (NT) saphenous vein (SV) grafts are superior to SVs harvested by the conventional technique (CT), with a patency comparable with the internal thoracic artery (ITA). Preservation of the vasa vasorum is implicated in the success of NT harvesting. We compared the vasa vasorum and endothelial nitric oxide synthase (eNOS) in NT SV with ITA and radial artery (RA) grafts. Skeletonized SV (SSV) was also analyzed. The NT SV had a higher number and larger vasa vasorum compared with ITA (P = .0001) and RA (P = .0004) that correlated with eNOS protein. Activity of eNOS in SSV grafts was significantly lower than NT SV grafts (P = 004). Since a high proportion of the vasa vasorum are removed in SSV using the CT, we suggest that preservation of the vasa vasorum and eNOS-derived NO contributes to the high patency for NT as compared with SSV grafts.

Endothelial nitric oxide synthase expression in postmenopausal women: a sex-specific risk factor in coronary surgery.

BACKGROUND: After coronary artery bypass graft surgery, older women have less favorable clinical outcome and lower conduit patency compared with men. This less favorable outcome can be in part ascribed to impaired endothelium-derived nitric oxide (eNOS) production. This study evaluated endothelial nitric oxide synthase expression in internal mammary artery from postmenopausal women undergoing coronary artery bypass graft surgery. METHODS: Internal mammary artery segments were obtained from 20 postmenopausal woman and 20 matched male patients. Twenty more segments from younger patients were used as controls. Expression of eNOS messenger RNA in internal mammary artery endothelial cells were evaluated by polymerase chain reaction and real-time quantitative reverse transcription polymerase chain reaction. The eNOS protein level was assayed by Western blot. Vascular dynamics of specimens were evaluated by organ chamber methodology. RESULTS: In postmenopausal women, the band of messenger RNA for eNOS was reduced by 37.4% and by 25.2%, respectively, compared with matched men and the control group (62.6%±4.8% versus 74.8%±5.3%, p<0.001). In comparison with the control group lane, the eNOS protein immunoreactive band was 44.2% decreased in postmenopausal women and 34.5% decreased in matched men, and was significantly decreased in postmenopausal women as compared with matched men (55.8%±4.6% versus 65.5%±5.2%, p<0.001). Nitric oxide-mediated vasomotor dynamics were consistent with reduced eNOS production. CONCLUSIONS: Internal mammary artery endothelial cells from women after menopause undergoing coronary surgery have impaired expression of messenger RNA for eNOS and reduced eNOS levels. Reduced bioactivity of nitric oxide translates into impaired endothelial metabolism that could contribute to worse surgical outcome.

Homocysteinylated protein levels in internal mammary artery (IMA) fragments and its genotype-dependence. S-homocysteine-induced methylation modifications in IMA and aortic fragments.

The resistance of internal mammary artery (IMA) toward atherosclerosis is not well understood. In plasma, homocysteine (Hcy) occurs in reduced, oxidized, homocysteine thiolactone and a component of proteins as a result of N- or S-homocysteinylation. We evaluated S/N-homocysteinylated protein levels in IMA fragments of patients undergoing coronary artery bypass grafting, and whether they were affected by genetic common variants. We tested whether tHcy, Hcy-S-protein levels, genotypes or Hcy-induced methylation modifications were related to differences in iNOS, Ddah2, and eNOS gene expression between territories. A small percentage of Hcy-S-proteins were found in IMA fragments. The Mthfr C677T (rs1801133) and Pon-1 Leu55Met (rs854560) variants were associated with Hcy-S-proteins. We observed a gradual difference according to Hcy-S-protein levels in the methylation degree of the Ddah2 gene promoter in aortic, but not in IMA, fragments. No correlation between the degree of methylation and the Ddah2 gene expression levels was found in both types of analyzed fragments. Total Hcy but not Hcy-S-proteins correlated with iNOS promoter methylation. Analyzed variants seem to contribute to the in vivo Hcy binding properties to IMA. The contribution of the Hcy-derived methylation modifications to Ddah2 and eNOS gene expression seems to be tissue-specific and independent of the Ddah2/ADMA/eNOS pathway. Hcy-derived methylation modifications to the iNOS gene promoter contribute to a lesser extent to iNOS gene expression.

Endothelial nitric oxide synthase enhancer for protection of endothelial function from asymmetric dimethylarginine-induced injury in human internal thoracic artery.

OBJECTIVES: Endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine is a cardiovascular risk factor that is elevated in patients with coronary artery disease. We hypothesized that novel endothelial nitric oxide synthase enhancer AVE3085 might improve the endothelial function altered by asymmetric dimethylarginine in the human internal thoracic artery. METHODS: Cumulative concentration-relaxation curves to acetylcholine (-11 to -5 log mol/L) were established in left internal thoracic artery rings (n = 65) from 27 patients undergoing coronary artery bypass grafting in precontraction induced by U46619 (-8 log mol/L) in the absence or presence of asymmetric dimethylarginine (100 µmol/L) or AVE3085 (30 µmol/L). Protein expressions of endothelial nitric oxide synthase and levels of superoxide anion production were detected. RESULTS: Maximal relaxation induced by acetylcholine was significantly attenuated by asymmetric dimethylarginine (12.7% ± 2.3% vs 35.3% ± 5.0% in control; P < .05) and significantly restored by AVE3085 (23.4% ± 2.8%; P < .05). AVE3085 also markedly restored endothelial nitric oxide synthase expression (0.29 ± 0.008; P = .012) reduced by asymmetric dimethylarginine (0.05 ± 0.04 vs 0.36 ± 0.03 in control; P = .014). Increased superoxide anion production by asymmetric dimethylarginine (2.97 ± 0.25 vs 0.51 ± 0.10 relative light units/[s/mg] in control; P < .05) was inhibited by AVE3805 (0.62 ± 0.104 relative light units/[s/mg]; P < .05). CONCLUSIONS: AVE3085 may restore endothelium-dependent relaxation reduced by asymmetric dimethylarginine through upregulation of endothelial nitric oxide synthase expression and inhibition of production of superoxide anion in human internal thoracic artery. These findings provide new insights into endothelial protection of coronary bypass grafting vessels to improve long-term patency of grafts.

Expression and function of endothelial nitric oxide synthase messenger RNA and protein are higher in internal mammary than in radial arteries.

BACKGROUND: The internal mammary artery (IMA) has a better long-term patency rate than the radial artery (RA), but the underlying molecular mechanisms are unclear. We compared endothelial nitric oxide synthase (eNOS) and related NO release in these two arteries. METHODS: Real-time polymerase chain reaction was used to quantify eNOS messenger RNA (mRNA) expression level in the endothelial cells of IMAs and RAs. eNOS protein localization was determined by immunohistochemistry. NO release from the endothelium of IMAs and RAs was directly measured by an electrochemical method using a membrane-type NO-sensitive electrode. RESULTS: Endothelial nitric oxide synthase mRNA expression level was significantly higher in the endothelial cells of IMAs than in RAs (1.03±0.19 vs 0.53±0.09, n=7, p<0.05), but was similar in the whole vascular tissue. eNOS protein immunoreactivity was higher in the endothelial cells of IMAs than in RAs. NO release at both levels in IMAs was significantly greater than in RAs (basal: 17.5±1.9 vs 10.2±0.7 nM, n=11 each, p=0.003; stimulated with bradykinin -7 log M: 31.5±3.6 vs 14.3±5.3 nM, n=6 each, p=0.02). CONCLUSIONS: Endothelial cells in the IMA express higher levels of eNOS mRNA and protein than those in the RA, which is linked with higher release of NO. These findings may be related to the superior long-term patency rate of the IMA vs the RA. This study also provides some basic genetic information for grafting arteries.

Dual actions of cilnidipine in human internal thoracic artery: inhibition of calcium channels and enhancement of endothelial nitric oxide synthase.

OBJECTIVE: Cilnidipine is a novel, long-action L/N-type dihydropyridine calcium channel blocker that has recently been used for antihypertensive therapy. We investigated the vasorelaxation effect of cilnidipine with regard to its calcium channel blockage and nitric oxide-cyclic guanosine monophosphate-dependent mechanism in human internal thoracic artery. METHODS: Fresh human internal thoracic arteries taken from discarded tissues of patients undergoing coronary artery bypass surgery were studied. Concentration-relaxation curves for cilnidipine in comparison with nifedipine were studied. The expression level of endothelial nitric oxide synthase mRNA was assayed by quantitative real-time polymerase chain reaction, and the phosphorylation of endothelial nitric oxide synthase at Ser(1177) was determined by Western blotting analysis. RESULTS: Cilnidipine and nifedipine caused nearly full relaxation in potassium-precontracted internal thoracic artery. Pretreatment with cilnidipine at the clinical plasma concentration significantly depressed the maximal contraction. Endothelium denudation (47.7% ± 7.0%, P < .05) and inhibition of endothelial nitric oxide synthase (48.6% ± 6.1%, P < .05) or guanylate cyclase (41.6% ± 3.8%, P < .01) significantly reduced the cilnidipine-induced endothelium-dependent relaxation (73.9% ± 6.4%). Cilnidipine increased the expression of endothelial nitric oxide synthase mRNA by 42.4% (P < .05) and enhanced phosphorylation level of endothelial nitric oxide synthase at Ser(1177) by 37.0% (P < .05). CONCLUSIONS: The new generation of calcium channel antagonist cilnidipine relaxes human arteries through calcium channel antagonism and increases production of nitric oxide by enhancement of endothelial nitric oxide synthase. The dual mechanisms of cilnidipine in human arteries demonstrated in this study may prove particularly important in vasorelaxing therapy in cardiovascular diseases.

Regulation of NO-dependent acetylcholine relaxation by K+ channels and the Na+-K+ ATPase pump in porcine internal mammary artery.

This study was designed to determine whether K+ channels play a role in nitric oxide (NO)-dependent acetylcholine relaxation in porcine internal mammary artery (IMA). IMA segments were isolated and mounted in organ baths to record isometric tension. Acetylcholine-elicited vasodilation was abolished by muscarinic receptor blockade with atropine (10(-6)M). Incubation with indomethacin (3 x 10(-6)M), superoxide dismutase (150 U/ml) and bosentan (10(-5)M) did not modify the acetylcholine response ruling out the participation of cyclooxygenase-derivates, reactive oxygen species or endothelin. The relaxation response to acetylcholine was strongly diminished by NO synthase- or soluble guanylyl cyclase-inhibition using L-NOArg (10(-4)M) or ODQ (3 x 10(-6)M), respectively. The vasodilation induced by acetylcholine and a NO donor (NaNO(2)) was reduced when rings were contracted with an enriched K+ solution (30 mM), by voltage-dependent K+ (K(v)) channel blockade with 4-amynopiridine (4-AP; 10(-4)M), by Ca(2+)-activated K+ (K(Ca)) channel blockade with tetraethylammonium (TEA; 10(-3)M), and by apamin (5 x 10(-7)M) plus charybdotoxin (ChTx; 10(-7)M) but not when these were added alone. In contrast, large conductance K(Ca) (BK(Ca)), ATP-sensitive K+ (K(ATP)) and inwardly rectifying K+ (K(ir)) channel blockade with iberiotoxin (IbTx; 10(-7)M), glibenclamide (10(-6)M) and BaCl(2) (3 x 10(-5)M), respectively, did not alter the concentration-response curves to acetylcholine and NaNO(2). Na+-K+ ATPase pump inhibition with ouabain (10(-5)M) practically abolished acetylcholine and NaNO(2) relaxations. Our findings suggest that acetylcholine-induced relaxation is largely mediated through the NO-cGMP pathway, involving apamin plus ChTx-sensitive K+ and K(v) channels, and Na+-K+-ATPase pump activation.

Short homologous peptides based on C-terminal sequences of fibrinogen ß- and γ-chains (Haptides) affect cardiovascular function by eNOS inhibition.

Haptides are a family of 19-21-mer cell-binding and permeating peptides homologous to sequences in the C termini on both fibrinogen ß- and γ-chain (Cß and preCγ, respectively). The effect of the Haptides on the cardiovascular system was studied by different assays, including the activity of isolated perfused rat heart and blood vessels in the organ bath. Haptides (50-80 µg/ml) decreased the hemodynamic functions of perfused rat hearts by up to 60% (p < 0.05) in a dose-dependent manner. Whole fibrinogen or a control nonrelated peptide (Cα) did not show such an effect. The NO donor, sodium nitroprusside, reversed the inhibitory effects of Haptides. L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, did not further augment the effect of the Haptides. Perfused (FITC)Haptides were attached to the coronary endothelium. In myocardial homogenates and HUVEC, Haptides significantly decreased eNOS activity, but had no effect on the contraction of isolated cultured adult cardiomyocytes. Haptides also significantly enhanced the contraction of rings of rat aorta and human mammary artery vessels ex vivo only when the endothelium was intact. Haptides seem to affect the coronary endothelium, but not the cardiomyocytes, by inhibiting eNOS activity, causing vasoconstriction, temporary ischemia and impaired myocardial function that seem to be related to the amino acid composition of the Haptides.

Increased RhoA/ROK mRNA expression and function in diabetic vein grafts compared with nondiabetic vein grafts and diabetic arterial grafts.

OBJECTIVE: Aim of the study was to discuss a new mechanism underlying the poor graft patency of GSV from diabetic patients and provide a rationale for selecting suitable grafts in diabetic patients. MATERIALS AND METHODS: The discarded matched RA, IMA, and GSV from 7 diabetics and 7 nondiabetic patients undergoing CABG were collected and tested for their contractile response to phenylephrine (PE) and their relaxation response to fasudil (a inhibitor of Rho-kinase) and used for immunohistochemical and mRNA detection of RhoA/ROK. RESULTS: The relaxation response to fasudil of GSV taken from diabetic patients was markedly increased but the relaxation response to fasudil of IMA and RA from diabetic patients was not. Immunohistochemistry and mRNA expression of RhoA/ROK was significantly increased in GSV from diabetic patients compared to that of IMA and RA from diabetic patients. RhoA/ROK immunohistochemistry and mRNA expression were significantly increased in GSV from diabetic patients compared with GSV from nondiabetic controls. CONCLUSIONS: RhoA/ROK expression and function in GSV from diabetic patients is significantly increased compared with IMA and RA from diabetic patients and GSV from nondiabetic patients. This contributes to a higher incidence of atherosclerosis and a lower long-term patency of GSV from diabetic patients.

Human internal thoracic arteries from diabetic patients are resistant to endothelial dysfunction.

The aim of this analysis was to compare vasoreactive properties of internal thoracic arteries (ITA) grafts from diabetic (DM) to those of non-diabetic (ND) patients. Ring segments of ITA, taken from patients undergoing coronary artery bypass grafting, were suspended in organ bath chambers filled with modified Krebs-Henseleit solution and contractile responses to potassium chloride (KCl), noradrenaline (NA), endothelin-1 (ET-l), and endothelium-dependent relaxant responses to acetylcholine (ACH) were recorded isometrically. The receptor-mediated agonists NA and ET-1 stimulated ITA from both groups within similar concentration ranges while ITA from DM patients proved to be significantly more sensitive to KCl than ITA from ND. Furthermore, maximal contractile responses indicated that KCl (3.79 +/- 0.30 g, n = 7 in DM and 2.50 +/- 0.23 g, n = 29 in ND, P < 0.05) evoked significantly higher responses in ITA from DM as compared to the ND control group while both NA and ET-l stimulated ITA from both groups with similar efficacies. Endothelium-dependent relaxant responses to ACH proved to be similar in both groups when expressed as percentages of the pre-existing tone. The present data support the contention that in comparison to ND controls arteries from DM patients are more sensitive to depolarization but endothelial dysfunction is less frequent in human ITA than expected from observations in systemic vascular beds.

Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways.

BACKGROUND: Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries. METHODS: Human internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists. RESULTS: The endothelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 microM bisindolylmaleimide I and 10 microM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 microM SB203580, 10 microM PD98059 and 10 microM SP600125, respectively). CONCLUSION: In conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.

Platelet-activating factor acetylhydrolase and transacetylase activities in human aorta and mammary artery.

Platelet-activating factor (PAF), the potent phospholipid mediator of inflammation, is involved in atherosclerosis. Platelet-activating factor-acetylhydrolase (PAF-AH), the enzyme that inactivates PAF bioactivity, possesses both acetylhydrolase and transacetylase activities. In the present study, we measured acetylhydrolase and transacetylase activities in human atherogenic aorta and nonatherogenic mammary arteries. Immunohistochemistry analysis showed PAF-AH expression in the intima and the media of the aorta and in the media of mammary arteries. Acetylhydrolase and transacetylase activities were (mean +/- SE, n = 38): acetylhydrolase of aorta, 2.8 +/- 0.5 pmol/min/mg of tissue; transacetylase of aorta, 3.3 +/- 0.7 pmol/min/mg of tissue; acetylhydrolase of mammary artery, 1.4 +/- 0.3 pmol/min/mg of tissue (P < 0.004 as compared with acetylhydrolase of aorta); transacetylase of mammary artery, 0.8 +/- 0.2 pmol/min/mg of tissue (P < 0.03 as compared with acetylhydrolase of mammary artery). Lyso-PAF accumulation and an increase in PAF bioactivity were observed in the aorta of some patients. Reverse-phase HPLC and electrospray ionization mass spectrometry analysis revealed that 1-O-hexadecyl-2 acetyl-sn glycero-3-phosphocholine accounted for 60% of the PAF bioactivity and 1-O-hexadecyl-2-butanoyl-sn-glycerol-3-phosphocholine for 40% of the PAF bioactivity. The nonatherogenic properties of mammary arteries may in part be due to low PAF formation regulated by PAF-AH activity. In atherogenic aortas, an imbalance between PAF-AH and transacetylase activity, as well as lyso-PAF accumulation, may lead to unregulated PAF formation and to progression of atherosclerosis.

ACE2 and AT4R are present in diseased human blood vessels.

A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.

Angiotensin-converting enzyme 2 (ACE2) expression and activity in human carotid atherosclerotic lesions.

Angiotensin-converting enzyme (ACE)2 is a recently identified homologue of ACE. As ACE2 inactivates the pro-atherogenic angiotensin II, we hypothesize that ACE2 may play a protective role in atherogenesis. The spatiotemporal localization of ACE2 mRNA and protein in human vasculature and a possible association with atherogenesis were investigated using molecular histology (in situ hybridization, immunohistochemistry). Also, the ACE : ACE2 balance was investigated using enzymatic assays. ACE2 mRNA was expressed in early and advanced human carotid atherosclerotic lesions. In addition, ACE2 protein was present in human veins, non-diseased mammary arteries and atherosclerotic carotid arteries and expressed in endothelial cells, smooth muscle cells and macrophages. Quantitative analysis of immunoreactivity showed that total vessel wall expression of ACE and ACE2 was similar during all stages of atherosclerosis. The observed ACE2 protein was enzymatically active and activity was lower in the stable advanced atherosclerotic lesions, compared to early and ruptured atherosclerotic lesions. These results suggest a differential regulation of ACE2 activity during the progression of atherosclerosis and suggest that this novel molecule of the renin-angiotensin system may play a role in the pathogenesis of atherosclerosis.

Insight into the role of CYBA A640G and C242T gene variants and coronary heart disease risk. A case-control study.

The CYBA gene variants have been inconsistently associated with coronary heart disease (CHD) risk. A case-control study was conducted genotyping 619 subjects to explore the contribution of C242T and A640G to CHD risk in the population. A significant risk was found associated with GG homozygosity (odds ratio (OR) 2.132, 95% confidence interval, 1.113-4.085). The C242T variant was associated with CHD risk in women. Bias due to population stratification was analysed. Phenotype changes linked to these polymorphisms were evaluated. Superoxide measurements revealed higher production as indicated by the presence of the G and T alleles. Differences in mRNA concentration in heterozygous A640G samples were analysed. Higher levels of G allele mRNA compared with A allele mRNA were found. NAD(P)H oxidase p22phox sub-unit expression was evaluated with Western blot. Experiments revealed a gradual relationship in p22phox protein expression according to genotypes of the analysed variants. Those GG TT double homozygous showed increased p22phox protein expressions regarding AA CC double homozygous. This study has demonstrated increased expression and activity of the NAD(P)H system components during atherogenesis and the results could help explain the relevance of the A640G variant as a CHD marker.

Increased matrix metalloproteinase 2 activity in the human internal mammary artery is associated with ageing, hypertension, diabetes and kidney dysfunction.

Dysregulation of matrix metalloproteinase (MMP)-2 in the vasculature has been suggested to be associated with increased prevalence of cardiovascular disease and renal injury. In this descriptive study, we hypothesized that arterial MMP-2 activity is elevated in the presence of cardiovascular risk factors such as diabetes, hypertension, smoking and ageing, and that it correlates with the degree of kidney function. MMP-2 activity in internal mammary arteries (n = 37) was measured using gelatinolytic zymography, and cutoffs were determined using sample-derived medians. Patient demographics and clinical data were analyzed, and the estimated glomerular filtration rate (eGFR) was calculated. High MMP-2 activity (>60,000 units) was associated with age, hypertension and diabetes (p = 0.0034, 0.06 and 0.0034, respectively). Multivariate analysis showed that age and diabetes were independent predictors of high MMP-2 activity. There is a trend towards increased MMP-2 activity and reduced eGFR (p = 0.010). The current exploratory work describes that the activity of MMP-2 in the internal mammary artery is correlated with age, hypertension, diabetes and eGFR. It is the first report suggesting that MMP-2 in the arterial vasculature could be the possible mediator crucial in linking the progression of kidney function to cardiovascular disease.