Co-Culture of Primary Human Coronary Artery and Internal Thoracic Artery Endothelial Cells Results in Mutually Beneficial Paracrine Interactions.

Although saphenous veins (SVs) are commonly used as conduits for coronary artery bypass grafting (CABG), internal thoracic artery (ITA) grafts have significantly higher long-term patency. As SVs and ITA endothelial cells (ECs) have a considerable level of heterogeneity, we suggested that synergistic paracrine interactions between CA and ITA ECs (HCAECs and HITAECs, respectively) may explain the increased resistance of ITA grafts and adjacent CAs to atherosclerosis and restenosis. In this study, we measured the gene and protein expression of the molecules responsible for endothelial homeostasis, pro-inflammatory response, and endothelial-to-mesenchymal transition in HCAECs co-cultured with either HITAECs or SV ECs (HSaVECs) for an ascending duration. Upon the co-culture, HCAECs and HITAECs showed augmented expression of endothelial nitric oxide synthase (eNOS) and reduced expression of endothelial-to-mesenchymal transition transcription factors Snail and Slug when compared to the HCAEC-HSaVEC model. HCAECs co-cultured with HITAECs demonstrated an upregulation of HES1, a master regulator of arterial specification, of which the expression was also exclusively induced in HSaVECs co-cultured with HCAECs, suggestive of their arterialisation. In addition, co-culture of HCAECs and HITAECs promoted the release of pro-angiogenic molecules. To conclude, co-culture of HCAECs and HITAECs results in reciprocal and beneficial paracrine interactions that might contribute to the better performance of ITA grafts upon CABG.

Platform technologies for decellularization, tunic-specific cell seeding, and in vitro conditioning of extended length, small diameter vascular grafts.

The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens.

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.

Vascular wall-resident CD44+ multipotent stem cells give rise to pericytes and smooth muscle cells and contribute to new vessel maturation.

Here, we identify CD44(+)CD90(+)CD73(+)CD34(-)CD45(-) cells within the adult human arterial adventitia with properties of multipotency which were named vascular wall-resident multipotent stem cells (VW-MPSCs). VW-MPSCs exhibit typical mesenchymal stem cell characteristics including cell surface markers in immunostaining and flow cytometric analyses, and differentiation into adipocytes, chondrocytes and osteocytes under culture conditions. Particularly, TGFß1 stimulation up-regulates smooth muscle cell markers in VW-MPSCs. Using fluorescent cell labelling and co-localisation studies we show that VW-MPSCs differentiate to pericytes/smooth muscle cells which cover the wall of newly formed endothelial capillary-like structures in vitro. Co-implantation of EGFP-labelled VW-MPSCs and human umbilical vein endothelial cells into SCID mice subcutaneously via Matrigel results in new vessels formation which were covered by pericyte- or smooth muscle-like cells generated from implanted VW-MPSCs. Our results suggest that VW-MPSCs are of relevance for vascular morphogenesis, repair and self-renewal of vascular wall cells and for local capacity of neovascularization in disease processes.

The effects of potassium channel opener P1075 on the human saphenous vein and human internal mammary artery.

Because adrenergic contractions can contribute to the development of life-threatening spasm of coronary artery bypass graft, this study was performed to investigate the effect of adenosine 3-phosphate (ATP)-sensitive K channel (KATP) opener P1075 on contractions of isolated human saphenous vein (HSV) and human internal mammary artery (HIMA). Phasic contractions were evoked by electric field stimulation (20 Hz) and noradrenaline. The sustained contractions were evoked by phenylephrine. The presence of pore-forming Kir6.1 and Kir6.2 subunits of the KATP channels in the HIMA and only Kir6.2 in the HSV was confirmed immunomorphologically. P1075 inhibited in the HSV only, the electrical field stimulation contractions more strongly than noradrenaline contractions. In addition, the phenylephrine contractions of HSV were more sensitive to P1075 in comparison to those of HIMA. Glibenclamide, a KATP channel blocker antagonized the vasodilatation produced by P1075 in both grafts differently, because its effect was more prominent on the P1075-induced inhibition of contractions of HSV than of HIMA. We conclude that P1075 has a vasorelaxant effect and inhibited adrenergic contractions of the tested grafts. This effect is graft and vasoconstrictor selective and seems to be mediated by Kir6.1- and/or Kir6.2-containing KATP channels. Thus, P1075 can be considered as a potential drug in the prevention of graft spasm.

Effect of amlodipine in human internal mammary artery and clinical implications.

BACKGROUND: Graft spasm remains challenging in CABG (coronary artery bypass grafting) surgery. We investigated the inhibitory effect of a dihydropyridine calcium antagonist amlodipine on the vasoconstriction mediated by potassium chloride (KCl), human urotensin-II (hU-II), and U46619 in human internal mammary artery (IMA) from patients undergoing CABG. METHODS: Isolated IMA rings (n = 78, taken from 42 patients) were studied in organ baths in two ways: the relaxing effect of amlodipine on vasoconstrictor-induced precontraction by KCl, hU-II, and U46619 and the depressing effect of amlodipine on the contraction. RESULTS: Amlodipine caused full relaxation in KCl-contracted (98.0% ± 2.1%), in hU-II-contracted (98.5% ± 2.4%), and in U46619-contracted (96.3% ± 1.3%) IMA rings (n = 8) with 15.5-fold higher potency to KCl than to hU-II (effective concentration causing 50% of maximal response [EC(50)]: -8.17 ± 0.28 vs -6.98 ± 0.01 log M, p < 0.001) and 19.5-fold that to U46619 (EC(50): -8.17 ± 0.28 vs -6.88 ± 0.08 log M, p < 0.001). Pretreatment of IMA with plasma concentrations of amlodipine (-6.6 log M) significantly depressed subsequent contraction to KCl (from 20.8 ± 2.5 mN to 7.6 ± 3.0 mN, p = 0.004) and hU-II (from 14.1 ± 4.2 mN to 3.8 ± 2.0 mN, p = 0.026), but did not significantly affect the contraction to U46619. CONCLUSIONS: We conclude that in human IMA amlodipine has a potent inhibitory effect on the vasoconstriction mediated by a variety of vasoconstrictors. Thus, use of amlodipine in CABG patients is favored in treating and preventing graft spasm.

Insulin-like growth factor-1 induces phosphorylation of PI3K-Akt/PKB to potentiate proliferation of smooth muscle cells in human saphenous vein.

Coronary revascularization by coronary artery bypass grafting (CABG) is recommended in patients with recurrent myocardial ischemia. However, the long-term results of CABG using saphenous vein (SV) graft, compared to internal mammary artery (IMA) graft, have not been satisfactory. The SV graft failure is due to the development of intimal hyperplasia, a process characterized by abnormal migration and proliferation of smooth muscle cells (SMCs) in the intimal layer of the vein graft. Insulin growth factor 1 (IGF-1) is a major mitogenic growth factor released at the site of the shear stress-induced graft injury. This study, for the first time, compares the extent of IGF-1-PI3K-Akt activation in isolated human bypass graft conduits. Human SV and IMA vessels were collected and SMCs isolated and cultured. In cultured SMCs, effect of IGF-1 was examined on total and phosphorylated PI3K, Akt and IGF-1R by Western blot analysis. Cell proliferation was measured using BrdU ELISA. There was no significant difference in the basal expression of phosphorylated PI3K, Akt and IGF-1R in SV and IMA SMCs from human bypass conduits. However, we observed an upregulation of IGF-1 receptors in the SV SMCs in response to IGF-1 stimulation with no effect in IMA SMCs. Furthermore, the immunoblotting and cellular activation of signaling ELISA (CASE) assay demonstrated a significantly higher activity of both PI3K and Akt in IGF-1-stimulated SV SMCs than IMA. This was inhibited by an IGF-1R blocking antibody. IGF-1 induced proliferation in both SV and IMA SMCs was inhibited by a PI3K inhibitor, wortmannin. These data demonstrate differential activity of IGF-1-induced PI3K-Akt activation, which was quantitatively and temporally greater in SV SMCs than in the IMA. This, at least in part, could explain the greater propensity of the SV conduits than the IMA to undergo intimal hyperplasia following CABG.

Altered beta-2 adrenergic receptor gene expression in human clinical hypertension.

OBJECTIVES: The beta-2 adrenergic receptor is involved in mediating vasodilatation via neurohumoral and sympathetic nervous system pathways. Alterations in beta-2 adrenergic receptor gene expression (mRNA transcription) may contribute to the hypertensive phenotype. Human gene expression in clinical phenotypes remains largely unexplored due to ethical constraints involved in obtaining human tissue. We devised a method to obtain normally discarded internal mammary artery tissue from coronary artery bypass graft patients. We then investigated differences in hypertensive and normotensive participants' beta-2 adrenergic receptor gene expression in this tissue. METHODS: We collected arterial tissue samples from 46 coronary artery bypass patients in a surgical setting. Using 41 of the samples, we performed TaqMan real-time polymerase chain reaction (RT-PCR) and used the delta delta cycle threshold (DeltaDeltaCt) relative quantitation method for determination of fold-differences in gene expression between normotensive and hypertensive participants. The beta-2 adrenergic receptor target was normalized to glyceraldehyde-phosphate dehydrogenase. RESULTS: Participants with hypertension had significantly less-expressed beta-2 adrenoceptor gene (2.76-fold, p<.05) compared to normotensive participants. After Bonferroni correction, gene expression did not differ by race, gender, type/dose of beta-blocker prescribed, positive family history of hypertension, or diagnosis of diabetes mellitus type 2. CONCLUSIONS: These data support the possibility of a molecular basis for impaired adrenoceptor-mediated vascular tone in hypertension. Modification and extension of this research is required.

Temporal PTEN inactivation causes proliferation of saphenous vein smooth muscle cells of human CABG conduits.

Abstract Internal mammary artery (IMA) coronary artery bypass grafts (CABG) are remarkably resistant to intimal hyperplasia (IH) as compared to saphenous vein (SV) grafts following aorto-coronary anastomosis. The reason behind this puzzling difference still remains an enigma. In this study, we examined the effects of IGF-1 stimulation on the PI3K-AKT/PKB pathway mediating proliferation of smooth muscle cells (SMCs) of IMA and SV origin and the specific contribution of phosphatase and tensin homologue (PTEN) in regulating the IGF-1-PI3K-AKT/PKB axis under these conditions. Mitogenic activation with IGF-1, time-dependently stimulated the phosphorylation of PI3K and AKT/PKB in the SV SMCs to a much greater extent than the IMA. Conversely, PTEN was found to be significantly more active in IMA SMCs. Transient overexpression of PTEN in SMCs of SV and IMA inhibited AKT/PKB activity and upstream of AKT/PKB, caused a reduction of IGF-1 receptors. Downstream, PTEN overexpression in SV SMCs induced the transactivation of tumour suppressor protein p53 by down-regulating the expression of its inhibitor MDM2. However, PTEN overexpression had no significant effect on MDM2 and p53 expression in IMA SMCs. PTEN overexpression inhibited IGF-1-induced SMC proliferation in both SV and IMA. PTEN suppression, induced by siRNA transfection of IMA SMCs diminished the negative regulation of PI3K-PKB signalling leading to greater proliferative response induced by IGF-1 stimulation. Thus, we show for the first time that early inactivation of PTEN in SV SMCs leads to temporally increased activity of the pro-hyperplasia PI3K-AKT/PKB pathway leading to IH-induced vein graft occlusion. Therefore, modulation of the PI3K-AKT/PKB pathway via PTEN might be a novel and effective strategy in combating SV graft failure following CABG.

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.

Prothrombotic gene expression profile in vascular smooth muscle cells of human saphenous vein, but not internal mammary artery.

BACKGROUND: The resistance of internal mammary artery (IMA) toward thrombotic occlusion and accelerated atherosclerosis is not well understood. This study analyzed gene expression profiles of vascular smooth muscle cells (VSMCs) from IMA versus saphenous vein (SV). METHODS AND RESULTS: 54'675 probe sets were examined by Affymetrix microarrays. Thirty-one genes belonged to the coagulation system; 2 were differentially expressed, namely tissue factor (TF) and tissue-type plasminogen activator (tPA). TF was 3.1-fold lower in IMA than SV (P=0.006), whereas tPA was 9.0-fold higher (P<0.001). TF mRNA expression was lower in IMA than SV (P<0.05); tPA was higher (P<0.001). TF protein expression was 4.2+/-0.5-fold lower in IMA than SV (P<0.001); tPA was 2.6+/-0.4-fold higher (P<0.01). In IMA VSMC supernatant, TF protein and activity was lower (P<0.05), TFPI and tPA protein higher (P<0.05 and P<0.005), and clotting time of human plasma prolonged (P<0.05) as compared to SV. Migration to TF/FVIIa (10(-9) mol/L) was 3-fold lower in IMA than SV (P=0.01); PAR-2 protein expression was similar (P=NS), PAR-2 blockade without effect (P=NS). CONCLUSIONS: Among the genes of the coagulation system, TF and tPA are differentially expressed in VSMCs from IMA versus SV. This is consistent with protection of IMA from thrombus formation and vascular remodeling.

Galectin-1, an endogenous lectin produced by arterial cells, binds lipoprotein(a) [Lp(a)] in situ: relevance to atherogenesis.

Lipoprotein(a) [Lp(a)], a modified LDL molecule, is implicated in atherogenesis. Mechanisms of the accumulation of [Lp(a)] in atherosclerotic vessels is lacking in literature. We sought to investigate the complementarities of the carbohydrate structures on Lp(a) and LDL with galectin-1(a carbohydrate binding protein) and whether endogenous galectin-1 binds Lp(a) in situ. We investigated T-antigen structures on Lp(a) and LDL by enzyme-linked lectin assay using T-antigen specific lectins, galectin-1 and jacalin. Both jacalin and galectin-1 bound strongly to Lp(a) and to a much lesser extent, to LDL. Galectin-1 recognition of the lipoproteins was abolished when the O-linked sugars were selectively removed. Localization of endogenous galectin-1 within histological sections of human internal mammary artery and in vitro binding of Lp(a) to the tissues was analyzed by immunohistochemical staining. The Lp(a)-binding pattern was found to overlap with the localization of galectin-1. The poor Lp(a)-binding on inhibiting tissue galectin-1 with lactose, suggested the binding of Lp(a) to galectin-1. This may be suggestive of a mechanism by which Lp(a) accumulates within arterial walls in atherogenesis.

Smooth muscle cells cultured from human saphenous vein exhibit increased proliferation, invasion, and mitogen-activated protein kinase activation in vitro compared with paired internal mammary artery cells.

OBJECTIVE: Smooth muscle cell (SMC) proliferation, invasion, and matrix metalloproteinase (MMP) secretion are key events in the development of intimal hyperplasia, the lesion that causes coronary artery bypass graft (CABG) failure. Saphenous vein (SV) grafts are the most commonly used bypass conduits but are markedly more susceptible to intimal hyperplasia than internal mammary artery (IMA) grafts. We hypothesized that this may be due to inherent functional differences between SV-SMCs and IMA-SMCs. In this study we used paired cultures of SV-SMCs and IMA-SMCs from the same patients and compared their rates of proliferation, invasion, migration, and MMP-2 and MMP-9 secretion. METHODS: SMCs were cultured from explants of paired SV and IMA from 22 patients undergoing CABG. SMC populations of equivalent passage were used to determine proliferation in response to 10% fetal calf serum (FCS), 10 ng/mL platelet-derived growth factor (PDGF), and 10 ng/mL basic fibroblast growth factor (bFGF) by counting cells during a 7-day period. Immunoblotting was used to quantify phosphorylation of p44/42-mitogen-activated protein kinase (MAPK). Invasion and migration rates of paired SMCs were quantified using a modified Boyden chamber technique in the presence or absence of a Matrigel basement membrane barrier (BD Biosciences, Oxford, UK). Conditioned media from invasion assays were analyzed for secretion of MMP-2 and MMP-9 by gelatin zymography. RESULTS: Analysis of areas under curves for 7-day proliferation assays revealed that the number of SV-SMCs in response to FCS, PDGF, and bFGF was 2.1, 2.0, and 2.3 times higher, respectively, than that of paired IMA-SMCs. Basal MAPK activation in SV-SMCs was approximately double that of paired IMA-SMCs. SV-SMCs exhibited a 2.1-fold increase in invasion rate (Matrigel barrier) compared with IMA-SMCs, but migration rates (no Matrigel barrier) and MMP-2 and MMP-9 secretion were similar for the two cell types. CONCLUSIONS: Human SV-SMCs are inherently more proliferative and invasive than paired IMA-SMCs, likely due to a relative increase in p44/42-MAPK activation. These inherent functional differences between SMC of different origins may contribute to the increased prevalence of intimal hyperplasia in SV grafts compared with IMA grafts.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway.

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease.

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

Human endothelial cells synthesize protein Z, but not the protein Z dependent inhibitor.

Protein Z (PZ) is a vitamin K-dependent protein isolated from human plasma, and acts as a cofactor for a serpin, called protein Z-dependent protease inhibitor (ZPI). A prothrombotic phenotype has been reported in PZ deficient mice, and PZ deficiencies have been observed in patients with arterial thrombotic events. PZ was immunologically detected in the endothelium of atherosclerotic arteries, suggesting that endothelial cells could be involved in the production of PZ. In this study we analyzed the synthesis and release of PZ and ZPI by human umbilical vein endothelial cells (HUVEC), representative of the macrovasculature, and by HMEC-1, a microvascular endothelial cell line. PZ was quantified by a specific ELISA in the supernatant and in the lysates of both cellular types. Western blotting of the supernatants showed the presence of a band of 62 kDa, identical to PZ synthesized by the hepatoma cell line HepG2. mRNA of PZ was also detected in each cellular type. PZ biosynthesis was unaffected by inflammatory cytokines in HUVEC, whereas a slight decrease of mRNA and PZ antigen (53.5 +/- 14.5% of protein synthesis as compared to the control, p < 0.01) and a modest increase (126 +/- 8.5% as compared to the control, p < 0.05) were induced respectively byTumor Necrosis Factor (TNF)-alpha (25 ng/ml) and oncostatin M (5 ng/ml) in HMEC-1. Immunological studies showed the presence of PZ near the nucleus and a possible expression of PZ at the membrane. In addition, PZ was present in the endothelial cells of both normal arterial and venous vessel sections. In contrast, neither ZPI nor its mRNA was detected in endothelial cells.

Vascular-wall remodeling of 3 human bypass vessels: organ culture and smooth muscle cell properties.

OBJECTIVES: Late graft occlusions after coronary artery bypass grafting have been ascribed to neointimal hyperplasia. Given the pivotal role of smooth muscle cells in the pathogenesis of neointimal hyperplasia and the phenotypic heterogeneity of smooth muscle cells across vessels, we hypothesized that differences in long-term graft patency are at least partly related to differences in smooth muscle cell properties. The aim of the present study was to compare the vascular-wall remodeling of human internal thoracic artery, radial artery, and saphenous vein bypass conduits. METHODS: We evaluated the intimal thickening of the human graft segments in organ cultures (histopathology, morphometric, and immunofluorescence analyses) and assessed the properties of cultured smooth muscle cells isolated from these vessels in terms of cell proliferation (tritiated thymidine incorporation), migration (modified Boyden chamber), and collagen synthesis (tritiated proline incorporation). RESULTS: The total vessel-wall growth index and the intimal growth index were significantly higher for saphenous vein rings than for radial artery and internal thoracic artery rings. Immunofluorescence analyses showed predominant involvement of smooth muscle cells in neointimal growth induced by organ culture of saphenous vein rings. Cell proliferation was significantly higher in saphenous vein smooth muscle cells than in radial artery smooth muscle cells and significantly higher in radial artery smooth muscle cells than in internal thoracic artery smooth muscle cells. Migration of smooth muscle cells from saphenous vein grafts was significantly greater than from internal thoracic artery or radial artery grafts. Collagen synthesis was similar in smooth muscle cells from internal thoracic artery, radial artery, and saphenous vein grafts. CONCLUSIONS: Ex vivo vascular-wall remodeling and smooth muscle cell intrinsic growth and migratory properties are dissimilar between arterial and venous grafts and might shed light on reported angiographic patency rates of these grafts.

Characterization of beta3-adrenoceptors in human internal mammary artery and putative involvement in coronary artery bypass management.

OBJECTIVES: The aim of the present study was to analyze whether beta3-adrenoceptors (beta3-ARs) were effectively present and functional in the human internal mammary artery (IMA). BACKGROUND: The beta1- and beta2-adrenoceptors classically mediate the relaxant effects of catecholamines in the vessels. In vitro and in vivo studies performed in various animal species described vasodilating effects due to activation of a third beta-ARs subtype (beta3). METHODS: Reverse transcription-polymerase chain reaction analysis, Western blot experiments, and pharmacological studies were carried out in human IMA samples harvested from 27 patients undergoing coronary bypass surgery. RESULTS: The beta3-ARs messenger ribonucleic acid and protein were detected in intact IMA, but were absent in endothelium-free samples. This finding was confirmed by immunohistochemical experiments. In organ baths, a beta3-AR agonist, SR 58611A, induced an endothelium-dependent relaxation of phenylephrine-precontracted IMA rings. This vasodilation was not modified by beta1/beta2-AR antagonists, but was greatly altered in the presence of L-748,337, a selective human beta3-AR antagonist. Moreover, the inhibition of nitric oxide (NO) synthases abolished the beta3-adrenergic vasodilation, suggesting the involvement of a NO-signaling pathway. CONCLUSIONS: Those results demonstrated the presence of beta3-ARs in the endothelial layer of human IMA. The present work highlights the role of beta3-ARs in vasomotor control of IMA and opens new fields of investigation in coronary bypass graft management, heart failure, and hypertension.