Variability in gelatinase expression in the walls of vessels used as aortocoronary conduits may impact long-term graft patency.

BACKGROUND: An imbalance between the activity of matrix metalloproteinases (MMPs), particularly gelatinases, and tissue inhibitors of metalloproteinases (TIMPs) is considered as one of the mechanisms leading to aortocoronary graft failure. AIM: We aimed to assess the variability in gelatinase expression in the walls of aortocoronary conduits and to evaluate its impact on coronary artery bypass grafting (CABG) outcomes. METHODS: The study included 101 consecutive patients (61 men and 40 women) who underwent CABG. An immunohisto-chemical analysis of MMP-2, MMP-9, TIMP-1, and TIMP-2 expression was performed on the cross-sections of the internal thoracic artery (ITA), radial artery (RA), and saphenous vein (SV). The histological findings were compared between patients with SV graft disease (SVGD[+] group) and those without occlusions in the SV (SVGD[-] group). RESULTS: The median MMP and TIMP expression was the weakest in the ITA wall. MMP expression was comparable between the RA and SV cross-sections, whereas TIMP expression was stronger in the RA than in the SV wall (p < 0.05). In most SV segments, but not in the arteries, immunostaining intensity for MMP was comparable to or stronger than for TIMPs. In the veins harvested from the SVGD(+) group, MMP-2 and MMP-9 tissue expression was more pronounced than in the SVGD(-) group. TIMP levels were comparable between groups. CONCLUSIONS: Imbalance in the metalloproteinase-to-inhibitor tissue expression in the vessel wall might predispose to graft failure. A stronger expression of TIMPs than MMPs in the arterial grafts might explain favourable long-term outcomes.

Activation of the mTORC1 pathway by inflammation contributes to vascular calcification in patients with end-stage renal disease.

BACKGROUND: Chronic inflammation plays an important role in the progression of vascular calcification (VC). This study was designed to explore the effects and underlying mechanisms of inflammation on VC in the radial arteries of patients with end-stage renal disease (ESRD) with arteriovenostomy. METHODS: Forty-eight ESRD patients were divided into control (n = 25) and inflammation groups (n = 23) according to plasma C-reactive protein (CRP) level. Surgically removed tissues from the radial arteries of patients receiving arteriovenostomy were used in this study. Alizarin Red S staining was used to examine calcium deposition. The expression of inflammation markers, bone structure-associated proteins and mammalian target of rapamycin complex1 (mTORC1) pathway-related proteins was assessed by immunohistochemical staining. RESULTS: The expression of tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) was increased in the radial arteries of the inflammation group. Additionally, Alizarin Red S staining revealed a marked increase in calcium deposition in the inflammation group compared to controls. Further analysis by immunohistochemical staining demonstrated that the deposition was correlated with the increased expression of bone-associated proteins such as bone morphogenetic proteins-2 (BMP-2) and osteocalcin and collagen I, which suggested that inflammation induces osteogenic differentiation in vascular tissues and that osteogenic cells are the main cellular components involved in VC. Interestingly, there was a parallel increase in the expression of phosphorylated mTOR (p-mTOR) and pribosomal protein S6 kinase 1 (p-S6K1) in the inflammation group. Furthermore, mTORC1 pathway-related proteins were significantly associated with the enhanced expression of bone formation biomarkers. CONCLUSIONS: Inflammation contributed to VC in the radial arteries of ESRD patients via the induction of osteogenic differentiation in vessel walls, which could be regulated by the activation of the mTORC1 pathway.

Activation of mTOR contributes to foam cell formation in the radial arteries of patients with end-stage renal disease.

BACKGROUND: Our previous in-vivo and in-vitro studies demonstrated that inflammation accelerated the progression of atherosclerosis via the dysregulation of the low-density lipoprotein receptor (LDLr) pathway. The current study aimed to investigate the effects and their underlying mechanisms of inflammation on lipid accumulation in the radial arteries of endstage renal disease (ESRD) patients with arteriovenostomy. METHODS: 30 ESRD patients with arteriovenostomy were included. The patients were divided into two groups based on their plasma levels of C-reactive protein: a control (n = 16) and an inflamed group (n = 14). The expression of tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein-1 of the radial arteries were increased in the inflamed group. Foam cell formation and lipid droplet accumulation were examined by hematoxylin and eosin (H & E) and Oil Red O staining. Intracellular cholesterol trafficking-related proteins were examined by immunohistochemistry and immunofluorescent staining. RESULTS: There was significant lipid accumulation in the radial arteries of the inflamed group compared with the control. Further analysis demonstrated that this accumulation was correlated with the increased protein expression of LDLr, sterol regulatory element-binding protein-2 (SREBP-2), and SREBP cleavageactivating protein (SCAP). Confocal microscopy showed that inflammation enhanced the translocation of SCAP escorting SREBP-2 from the endoplasmic reticulum to the Golgi, thereby activating LDLr gene transcription. Interestingly, upregulated LDLr expression was positively associated with the increased protein expression of mammalian target of rapamycin (mTOR), which had enhanced coexpression with SREBP-2. This finding suggests that the activation of mTOR may be involved in LDLr pathway disruption through the upregulation of SREBP-2 expression. CONCLUSION: Inflammation contributed to foam cell formation in the radial arteries of ESRD patients via the dysregulation of the LDLr pathway, which could be modulated by the activation of the mTOR pathway.

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.

Proteomic identification of matrix metalloproteinase substrates in the human vasculature.

BACKGROUND: Matrix metalloproteinases (MMPs) play a key role in cardiovascular disease, in particular aneurysm formation and plaque rupture. Surprisingly, little is known about MMP substrates in the vasculature. METHODS AND RESULTS: We used a proteomics approach to identify vascular substrates for 3 MMPs, 1 of each of the 3 major classes of MMPs: Human arteries were incubated with MMP-3 (a member of stromelysins), MMP-9 (considered a gelatinase), and MMP-14 (considered a member of the collagenases and of the membrane-bound MMPs). Candidate substrates were identified by mass spectrometry based on increased release from the arterial tissue on digestion, spectral evidence for proteolytic degradation after gel separation, and identification of nontryptic cleavage sites. Using this approach, novel candidates were identified, including extracellular matrix proteins associated with the basement membrane, elastic fibers (emilin-1), and other extracellular proteins (periostin, tenascin-X). Seventy-four nontryptic cleavage sites were detected, many of which were shared among different MMPs. The proteomics findings were validated by immunoblotting and by digesting recombinant/purified proteins with exogenous MMPs. As proof-of-principle, results were related to in vivo pathology by searching for corresponding degradation products in human aortic tissue with different levels of endogenous MMP-9. CONCLUSIONS: The application of proteomics to identify MMP targets is a new frontier in cardiovascular research. Our current classification of MMPs based on few substrates is an oversimplification of a complex area of biology. This study provides a more comprehensive assessment of potential MMP substrates in the vasculature and represents a valuable resource for future investigations.

Expression of both matrix metalloproteinase-2 and its tissue inhibitor-2 in tunica media of radial artery in uremic patients.

OBJECTIVE: To investigate the expression and clinical significance of both matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (tissue inhibitor of metalloproteinase-2 (TIMP-2)) in tunica media of radial artery in uremic patients. METHODS: The modified radial arteries from 80 uremic patients during internal arteriovenous fistula surgery were selected and used as experimental specimens. The calcification of tunica media was observed by alizarin red staining, and the expression status of MMP-2, TIMP-2, osteoprotegerin (OPG), and osteopontin (OPN) in tunica media of radial arteries of these patients was detected by immunohistochemical method. The semiquantitative analysis and comparison were conducted based on the calcification grading and the expression of each test protein in tunica media of radial artery. RESULTS: Of the 80 cases of radial artery specimens, 37 cases were presented with various degrees of calcification of tunica media, and the calcification rate was 46.25%; the expression of MMP-2, TIMP-2, OPG, and OPN could be detected in the calcificated tunica media of the radial artery and was positively correlated with the degree of vascular calcification (p < 0.05). CONCLUSION: The incidence of vascular calcification in uremic patients was high. The occurrence of calcification in tunica media of the radial artery was correlated with the expression of MMP-2 and TIMP-2.

Differences in angiotensin convertase enzyme (ACE) activity and expression may contribute to shorter event free period after coronary artery bypass graft surgery.

AIMS: The goal of this study was to investigate the importance of the vascular angiotensin convertase enzyme (ACE) in coronary artery bypass graft surgery (CABG) patients. METHODS: Vascular tissue (distal saphenous vein [n= 163] and/or radial artery [n= 120] segments) and blood samples were collected from CABG patients (n= 81). We studied (i) the potency of angiotensin I (AngI) and angiotensin II (AngII) to evoke vascular contractions; (ii) vascular and plasma ACE concentrations; and (iii) ACE genotype of the patients enrolled. RESULTS: The ratio of the potencies (EC(50) ) of AngII and AngI was significantly lower in radial artery compared to the saphenous vein (0.17 ± 0.03 nM and 0.51 ± 0.14 nM, respectively, P= 0.003), suggesting a 3-fold more effective AngI conversion in saphenous vein samples. Angiotensin constrictions were inhibited with telmisartan and captopril in both saphenous veins and radial arteries. Vascular ACE expression was significantly higher in saphenous vein compared to radial artery (9.7 ± 1.0 ng/mg and 5.3 ± 0.7 ng/mg, respectively, P= 0.01). Serum but no tissue ACE concentration was determined by ACE insertion/deletion polymorphism. Accordingly, no relation was found between serum and tissue ACE expression. CONCLUSION: ACE-inhibitor therapy targeting tissue located ACE may be beneficial to patients with saphenous vein grafts after CABG surgery.

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.

[The morphometry and eNOS expression of radial artery in elderly patients with coronary atherosclerotic heart disease].

OBJECTIVE: To compare the morphometry and endothelial nitric oxide synthase (eNOS) expression of radial artery (RA) between young and elderly patients with coronary atherosclerotic heart disease. METHODS: From February 2008 to June 2009, 219 patients underwent coronary artery bypass grafting (CABG) with autologous RA, 57 patients aged beyond 70 years and 64 patients aged under 60 years. Before RA was harvested, a modified Allen test was routinely performed. If positive, RA would be further evaluated with Doppler ultrasound examination. In both groups RA was collected for HE staining to evaluate percentage of luminal narrowing (LN) and relationship between intima and media width at maximum intimal thickness (IMR). Immunofluorescence and Western blot were used to investigate the location and expression level of eNOS within the wall of RA. RESULTS: Morphometry of RA in both young and elderly patients represented mild or moderate intimal hyperplasia, and medial calcification was not found. LN in elderly patients was (22 ± 6)%, while in young patients, it was (23 ± 6)%. IMR in elderly patients was 0.36 ± 0.21, while in young patients, it was 0.42 ± 0.19. There was no significant difference in both LN and IMR between two groups (P > 0.05). Immunofluorescence indicated RA in both groups revealed a high expression of eNOS in intima and media, particularly in the smooth muscle of media. The values of relative integrated optical density in elderly patients was 1.21 ± 0.13, while in young patients, it was 1.25 ± 0.12. Also there was no significant difference in the expression level of eNOS within the wall of RA (P > 0.05). CONCLUSION: After preoperative assessment with modified Allen's test and Doppler analysis, RA used as graft in the elderly has similar quality and function with young patients, and it may lead to a high patency in long term.

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.

Rho-kinase inhibitors Y-27632 and fasudil prevent agonist-induced vasospasm in human radial artery.

Radial artery (RA) vasospasm remains a potential cause of early graft failure after coronary artery bypass graft surgery, despite pretreatment with alpha-adrenergic or calcium channel blockers. Our aim was to investigate the mechanism of the vasorelaxant effects of Rho-kinase inhibitors (Y-27632 and fasudil) on the human RA. Segments were obtained from 30 patients undergoing coronary artery bypass graft and were divided into 3-4 mm vascular rings. The rings were stimulated with 10(-5) mol/L phenylephrine (PE) by using the isolated tissue bath technique and were relaxed with 10(-6) mol/L acetylcholine. Relaxation responses were recorded for Y-27632 (10(-9)-10(-4) mol/L), fasudil (10(-9)-10(-4) mol/L), and sodium nitroprusside (SNP) (10(-9)-10(-5) mol/L). Y-27632 and fasudil relaxation responses were repeated in either N(G)-nitro-L-arginine (L-NNA), which is a specific endothelial nitric oxide synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which is a guanylate cyclase inhibitor. SNP relaxation responses were repeated in 10(-8) mol/L Y-27632 and 10(-8) mol/L fasudil. Y-27632 and fasudil caused concentration-dependent vasorelaxation in RA rings precontracted with PE, and maximal relaxation (100%) was recorded at the highest concentration used (10(-4) mol/L). The vasorelaxant effects of Y-27632 and fasudil were significantly reduced in the presence of L-NNA and ODQ, and the pD2 values of Y-27632 and fasudil were not changed. The vasorelaxant effects of SNP were significantly increased in the presence of Y-27632 and fasudil, and the pD(2) values of SNP were not changed. These findings indicate that Y-27632 and fasudil caused concentration-dependent vasorelaxation in the RA rings. Because this effect was decreased in a dose-dependent manner by L-NNA and ODQ, the relaxant effects of Y-27632 and fasudil could be due to stimulation by nitric oxide that is being released. Rho-kinase inhibitors may have an important role in preventing vasospasm in arterial grafts used for coronary artery surgery.

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.

Localization of nitric oxide synthase type III in the internal thoracic and radial arteries and the great saphenous vein: a comparative immunohistochemical study.

BACKGROUND: Endothelial nitric oxide synthase type III is the key enzyme of the nitric oxide production in the vessel wall. In this study the localization of endothelial nitric oxide synthase type III within the wall of the human internal thoracic and radial arteries and the great saphenous vein was investigated. METHODS: Specimens were harvested from 23 patients undergoing surgical myocardial revascularization and submitted to light and electron microscope analysis using histochemical stainings and immunohistochemistry with specific antibodies anti-endothelial nitric oxide synthase type III, Factor VIII, and alpha-smooth muscle actin. RESULTS: Endothelial nitric oxide synthase type III was evident in the intima of all conduits and, unexpectedly, in the muscle cells of the media of muscular internal thoracic arteries and radial arteries. No endothelial nitric oxide synthase type III expression was found in the media of great saphenous veins. Semiquantitative analysis revealed a higher endothelial nitric oxide synthase type III expression in the wall of internal thoracic artery, particularly at the level of the media. CONCLUSION: Endothelial nitric oxide synthase type III is expressed in the intima of the internal thoracic and radial artery and the great saphenous vein and in the muscle cells of the media of the internal thoracic and radial arteries. However, the internal thoracic artery shows a higher intensity of endothelial nitric oxide synthase type III expression, particularly within the media. The present study provides the first demonstration of the endothelial nitric oxide synthase type III expression at the level of the smooth muscle cells of the tunica media of systemic human arteries and can provide an histologic explanation for the better results of the internal thoracic artery when used for coronary artery bypass grafting.

Expression and function of angiotensin converting enzyme, chymase, and angiotensin II in the human radial artery and internal thoracic artery.

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.