Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids.

BACKGROUND AND PURPOSE: Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids. EXPERIMENTAL APPROACH: Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca2+ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells. KEY RESULTS: Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1-2min. Dehydroepiandrosterone sulphate or 17ß-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid. CONCLUSIONS AND IMPLICATIONS: Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.

Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein.

AIMS/HYPOTHESIS: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide. METHODS: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance. RESULTS: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days). CONCLUSIONS/INTERPRETATION: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.

Diabetes and the abdominal aortic aneurysm.

OBJECTIVE: The aim of this review is to delineate the association between abdominal aortic aneurysms (AAAs) and diabetes mellitus. Mechanisms for the underlying association are then discussed. METHODS: A systematic review of the English-language literature using PubMed, EMBASE and Cochrane databases was undertaken up to September 2009. Studies reporting appropriate prevalence data were identified and a meta-analysis performed. RESULTS: Eleven studies were identified. The prevalence of diabetes mellitus in studied patients with AAA ranged from 6% to 14%. The prevalence of diabetes in control patients without AAA ranged from 17% to 36%. Pooled analysis suggested a reduced rate of diabetes amongst people with AAA compared to those without (OR 0.65, 0.60-0.70, p<0.001). CONCLUSIONS: Studies so far suggest a protective role for diabetes on the development of AAA. Further research is required to demarcate the underlying mechanisms for this possible association.

Characterisation of fractalkine/CX3CL1 and fractalkine receptor (CX3CR1) expression in abdominal aortic aneurysm disease.

OBJECTIVES: Fractalkine (CX3CL1) promotes adhesion and extravasation of leucocytes through interactions with fractalkine receptor (CX3CR1) expressed on CD56+/CD16+ NK cells and CD8+ T cells. The current study aims to test the hypothesis the CX3CL1-CX3CR1 interaction contributes to the inflammatory infiltrate in AAA tissue. DESIGN AND METHODS: Immunohistochemistry (IHC) was used to define expression of CX3CR1 in AAA tissue. Multi-parametric flow cytometry (FC) was used to determine CX3CR1 expression on T-cells (CD3+) and NK cells (CD56+) from AAA tissue and peripheral blood of AAA patients and healthy controls. Regulation of CX3CL1 expression by vascular endothelial (vEC) and smooth muscle cells (vSMC) was examined in vitro using primary cell cultures. RESULTS: CX3CR1+ cells were detected in 19/28 AAA tissue samples and predominately localised in the adventitia. PBMCs from patients with AAA demonstrated higher percentages of CX3CR1+ NK cells (60.0-88.6%) and T cells (7.5-39.4%) compared with healthy controls. Furthermore, the frequency of CX3CR1+ NK cells (91%) and T cells (94%) in inflammatory AAA tissue were higher than in atherosclerotic AAA tissue. The pro-inflammatory cytokine TNFalpha increased expression of fractalkine by vSMC and vEC. CONCLUSION: CX3CL1+ and CX3CR1+ cells are present in AAA disease and their interaction may contribute to the recruitment of inflammatory cells seen in AAA tissue.

Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

A comparison of six statins on the development of intimal hyperplasia in a human vein culture model.

OBJECTIVE: Intimal hyperplasia (IH) threatens the patency of up to 35% of saphenous vein (SV) bypass grafts. In addition to reducing cholesterol levels, statins may modulate smooth muscle cell proliferation and migration. Statins inhibit matrix metalloproteinase (MMP) activity. We therefore investigated the effect of six statins on neointimal formation and MMP activity in human SV organ culture. STUDY DESIGN: Human SV specimens were cultured for 14 days in the presence of six different statins and subsequently processed for measurement of neointimal thickness and MMP activity. The drug concentrations chosen corresponded to the manufacturers' Cmax. RESULTS: The six statins all significantly reduced IH development (P = 0.004) in association with reduced expression of proMMP-2 and 9 (P = 0.03) and reduced activity of activated MMP-2 and 9 (P = 0.03). CONCLUSION: This study suggests that the potential benefit of statins in reducing IH is a class effect and not confined to specific statins. The reduction of IH produced by statins may in part be due to their inhibition of MMPs.

Enhanced invasive properties exhibited by smooth muscle cells are associated with elevated production of MMP-2 in patients with aortic aneurysms.

BACKGROUND: abdominal aortic aneurysms (AAA) are associated with excessive vascular matrix remodelling. Recent findings suggest a systemic overproduction of matrix metalloproteinases-2 (MMP-2) by vascular smooth muscle cells (SMC) may be pivotal aetiologically. SMC migration is facilitated by MMP mediated proteolysis of the basement membrane and extracellular matrix. Our aim was to see if enhanced MMP-2 production by these SMC exhibit increased invasion, in an in vitro model of migration. METHOD: SMC were derived from inferior mesenteric vein (IMV) harvested from patients undergoing aneurysm repair (n=6) or colectomy for diverticulosis (n=6, control). Using a modified Boyden chamber chemotaxis was measured towards platelet derived growth factor (PDGF) and foetal calf serum (FCS) and invasion through a Matrigel layer. MMP-2 production was quantified by ELISA and gelatin zymography. RESULTS: chemoattractant studies demonstrated no difference in the effect of PDGF or FCS between the two populations of SMC. However, invasive studies demonstrated a significant increase in the number of migrating SMC isolated from IMV of AAA patients. Analysis of culture media extracts revealed that this difference was associated with a significant increase in production of MMP-2. CONCLUSION: SMC derived from patients with AAA demonstrate increased invasive properties when compared to a control group. Increased migration appears to be due to overproduction of MMP-2. The enhanced migratory potential of these SMC may lead to extracellular matrix remodelling and subsequent medial disruption demonstrated in the aneurysmal aorta. These data further support evidence of the proteolytic role of MMP-2 in cell migration.

Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9.

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.

The role of big endothelin-1 in colorectal cancer.

INTRODUCTION: Changes in liver blood flow caused by an unknown splanchnic vasoconstrictor have been noted in colorectal cancer patients with liver metastases. This prospective study was performed to assess whether plasma levels of big endothelin-1 (big ET-1) were raised in patients with colorectal cancer. METHODS: Plasma samples from peripheral vein of patients who underwent surgery for primary colorectal cancer (n=60) and those with known colorectal liver metastases (n=45) for a period of 15 months were taken prior to treatment and compared to age- and sex-matched controls (n=20). Plasma samples were analysed by using a single-step sandwich enzyme immunoassay. Immunohistochemistry and in situ hybridisation were also performed on tumour sections to investigate the expression of ET-1 by cancer cells. RESULTS: The median (range) plasma concentration of big ET-1 in controls was 2.1 pg/mL (1.2-13.4 pg/mL). The median (range) plasma concentration of big ET-1 in colorectal cancer patients with no overt hepatic metastases was 3.8 pg/mL (1.2-15.8 pg/mL), p=0.002, and the median (range) plasma concentration of big ET-1 in colorectal cancer patients with hepatic metastases was 5.2 pg/mL (1.7-30 pg/mL), p=0.0001; both were significantly elevated compared to the control group. A significant difference in immunostaining for big ET-1 was noted between paired normal colonic mucosa (median score-1) and tumour sections (median score-3), p=0.01. CONCLUSION: This study has demonstrated elevated concentrations of big ET-1 in colorectal cancer patients, especially in those with hepatic metastases. Upregulation of ET activity in colorectal cancer could be inferred by the increased immunostaining of big ET-1 in cancer cells. Therefore, plasma big ET-1 levels should be evaluated as a potential tumour marker for the identification of hepatic metastases at an earlier stage.

Inhibition of neointima formation in an organ culture of human saphenous vein: a comparison of dual endothelin-converting enzyme/neutral endopeptidase and selective neutral endopeptidase inhibition.

PURPOSE: Endothelin-1 (ET-1) has been implicated in a variety of vascular pathologic conditions, although there is considerable controversy as to whether such effects are mediated by the ET-(A) or ET-(B) receptor. This study investigated whether inhibition of big ET-1 processing by inhibition of endothelin-converting enzyme (ECE) could, therefore, offer an alternative therapeutic strategy in the prevention of vein graft intimal hyperplasia. METHODS: Human saphenous vein (3 equal segments from 10 patients) were maintained in organ culture for 14 days with either 50 micromol/L CGS 26303 (a dual ECE/neutral endopeptidase [NEP] inhibitor), 50 micromol/L CGS 24592 (a selective NEP inhibitor), or vehicle (control). They were then processed for immunostaining and neointimal thickness measurements, and conditioned media was collected for enzyme-linked immunosorbent assay analysis. RESULTS: Neointimal thickness in the ECE/NEP-inhibited veins did not differ significantly from that of control segments. However, there was a highly significant augmentation in the NEP-inhibited segments, consistent with an inhibition of ET-1 degradation (median difference, 16.8; 95% CI, -23.5, -10.4; P =.002, Wilcoxon). ECE immunostaining was reduced in the ECE/NEP-inhibited veins, although ET-1 staining was also present. ET-1 expression was intense in the thickened neointimas of NEP-inhibited veins, which also showed significant ECE staining. Elevated levels of big ET-1 were measured in the ECE/NEP-inhibited veins, consistent with reduced ECE activity. However, mature ET-1 was still detectable in these segments. CONCLUSION: There is a requirement for potent and selective inhibitors of ECE to evaluate fully the potential therapeutic benefits of blocking ET-1 biosynthesis. The use of dual inhibitors complicates the interpretation of results, because the observed response is likely to be a combination of both ECE and NEP inhibition.

Antiproliferative effect of UTP on human arterial and venous smooth muscle cells.

We have investigated the hypothesis that responses associated with proliferation are regulated by extracellular nucleotides such as ATP and UTP in cultured human vascular smooth muscle cells (VSMC) derived from internal mammary artery (IMA) and saphenous vein (SV). Platelet-derived growth factor (PDGF), ATP, and UTP each generated an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in both IMA- and SV-derived cells in the absence of detectable inositol 1,4,5-trisphosphate production. ATP alone had no effect on [(3)H]thymidine incorporation into DNA, but with a submaximal concentration of PDGF it raised [(3)H]thymidine incorporation in SV- but not IMA-derived cells. UTP alone also was without effect on [(3)H]thymidine incorporation or cell number. However, in both SV- and IMA-derived cells, UTP reduced the PDGF-stimulated [(3)H]thymidine response and PDGF-stimulated cell proliferation. This cannot be explained by an inhibitory effect on the p42/p44 mitogen-activated protein kinase (MAPK) cascade, since this response to PDGF was not attenuated by UTP. We conclude that, in human VSMC of both arterial and venous origin, UTP acts as an anti-proliferative regulator.

Raised levels of plasma big endothelin 1 in patients with colorectal cancer.

BACKGROUND: The aim was to assess the role of plasma Big Endothelin (ET) 1 levels as a marker of disease presence and stage in colorectal adenocarcinoma. METHODS: Big ET-1 was measured in the plasma of 37 patients with colorectal cancer. Preoperative systemic plasma levels of Big ET-1 in patients with cancer were compared with levels in 20 age- and sex-matched controls. Portal plasma samples were collected at operation in addition to peripheral venous samples. Immunohistochemical staining for Big ET-1 was performed on a selection of primary tumour specimens and liver metastases. RESULTS: Median (range) preoperative systemic plasma levels of Big ET-1 were significantly higher in patients with cancer than in controls (1.0 (0.3-9.7) versus 0.2 (0.0-6.0) fmol/ml; P = 0.0001). Intraoperative portal plasma levels of Big ET-1 were significantly higher in patients with Dukes' 'D' disease than in patients with Dukes' A, B and C disease (2.1 (1.4-10.0) versus 1.2 (0.3-6.6) fmol/ml; P = 0. 01). Similarly, systemic plasma levels were significantly higher in patients with Dukes' 'D' disease than in those with localized disease (1.9 (1.2-9.7) versus 1.2 (0.2-8.3) fmol/ml; P = 0.01). The presence of microvascular invasion in the tumour specimens was associated with a significantly raised portal plasma level of Big ET-1 (1.6 (1.5-2.1) versus 1.1 (0.8-1.3) fmol/ml; P = 0.04). Immunohistochemistry localized Big ET-1 to the cancer epithelial cells. CONCLUSION: The plasma level of Big ET-1 is significantly raised in patients with colorectal cancer. Patients with liver metastases have significantly higher levels than those with localized disease.

Marimastat inhibits neointimal thickening in a model of human arterial intimal hyperplasia.

OBJECTIVE: matrix metalloproteases (MMPs) produced by vascular smooth-muscle cells (VSMCs) degrade extracellular matrix and facilitate the migration of these cells. This is a fundamental process in arterial intimal hyperplasia. This study investigated whether Marimastat (a selective but non-specific MMP inhibitor) can prevent intimal hyperplasia in cultured human internal mammary artery (IMA). MATERIALS AND METHODS: segments of IMA from 8 patients were prepared and cultured for 14 days in serum-supplemented medium (control) or in medium supplemented with Marimastat at 2 concentrations (treatment groups). The tissue was fixed, sectioned, stained and neointimal thicknesses measured by computer-aided image analysis. Further sections were cultured in the same manner and prepared for gel enzymography to quantify the production of MMPs. RESULTS: neointimal thickness was significantly reduced by Marimastat in a dose-dependent manner when compared to controls (p =0.008 Wilcoxon). Gel enzymography demonstrated a reduction in levels of MMP2 and MMP9. This was most significant for the active forms of the enzymes ( p =0.03). CONCLUSIONS: our results suggest that there is a potential therapeutic role for specific inhibition of the gelatinases in the prevention of human arterial restenosis.

Production and inhibition of the gelatinolytic matrix metalloproteinases in a human model of vein graft stenosis.

OBJECTIVES: human vein graft stenoses are caused by intimal hyperplasia, a process which is characterised by extensive degradation and accumulation of extracellular matrix. This study investigated the role of the matrix metalloproteinases (MMPs) - the principal physiological mediators of extracellular matrix degradation - in the development of intimal hyperplasia in cultured human long saphenous vein. DESIGN: experimental study. MATERIALS AND METHODS: paired venous segments with the endothelium intact or denuded were cultured in standard conditions for 14 days. At the termination of culture, MMPs were extracted from one half of the tissue, whilst the remainder of the vein was prepared for histological examination. RESULTS: stereologic analysis revealed that the endothelium intact veins developed a significantly thicker neointima when compared to the denuded venous segments (20 micron v. 0 micron, p=0.006). Quantification of MMPs by substrate gel enzymography demonstrated that the development of a neointima was associated with increased production of the gelatinolytic MMP-9 (p=0. 03) in intact veins. Immunocytochemistry showed that the MMP-9 localised to the internal elastic lumina, which suggested a role in facilitating smooth-muscle-cell migration into the intima. The role of MMPs-2 and -9 in intimal hyperplasia was further investigated by culturing intact venous segments with a therapeutic concentration of doxycycline--a potent MMP inhibitor. These experiments demonstrated that a therapeutic dose of doxycycline significantly reduced neointimal thickness (control 21 micron, doxycycline 10 mg/l-5.5 micron), in conjunction with a significant reduction in the production of MMP-9. CONCLUSIONS: these data suggest that elevated levels of MMPs may play a significant role in the development of human intimal hyperplasia and that inhibition of these enzymes may offer a potential therapeutic strategy for the prevention of hyperplastic lesions.

Endothelin-B receptors mediate intimal hyperplasia in an organ culture of human saphenous vein.

OBJECTIVE: Although a number of pharmacologic agents have been shown to reduce intimal hyperplasia in animal models of restenosis, to date no systemic agent has conclusively been shown to be effective in humans. Recently, considerable attention has been directed towards endothelin (ET), a potent vasoconstrictor and a powerful mitogen for vascular smooth muscle cells, as a mediator of intimal hyperplasia. Endothelin-1 has been shown to be mitogenic for human saphenous vein smooth muscle cells, and expression also is elevated in human vein graft stenosis. The aim of this study was the investigation of whether ET receptor antagonists can attenuate neointima formation in a laboratory model of vein graft intimal hyperplasia and the determination of whether the effects are mediated by a specific ET receptor subtype. METHODS: We used an organ culture of human saphenous vein, a well-validated model of vein graft intimal hyperplasia. Paired segments of human long saphenous vein were cultured with and without the following antagonists: bosentan, a nonselective ET receptor antagonist; BQ 123, a specific endothelin-A antagonist; or BQ 788, a specific endothelin-B (ETB) antagonist. After 14 days in the culture, the segments were fixed and processed and the sections were immunostained to facilitate the measurements of neointimal thickness with a computerized image analysis system. RESULTS: The nonselective antagonist bosentan and the ETB selective antagonist BQ 788 significantly reduced neointima formation by 70% (P = .001) and 50% (P = .03), respectively, but the ETA antagonist BQ 123 had no significant effect on the reduction of neointima formation (P = 1.0). CONCLUSION: The results of this study imply an important role for ET as a mediator of human vein graft intimal hyperplasia and imply further that a specific ETB antagonist may have a therapeutic potential for the prevention of vein graft stenosis.

Marimastat inhibits neointimal thickening in a model of human vein graft stenosis.

BACKGROUND: There is now accumulating evidence that matrix metalloproteinases (MMPs), the physiological mediators of matrix deposition and degradation, play an important role in the development of intimal hyperplasia following arterial bypass. This study investigated the effect of marimastat, an orally active specific MMP inhibitor, on neointima formation in cultured human saphenous vein. METHODS: Segments of human saphenous vein obtained from ten patients undergoing arterial bypass surgery were cultured for 14 days in serum-supplemented RPMI medium (controls) or in control medium supplemented with marimastat at three different concentrations (treatment groups). Following culture, half of each segment was prepared for histological examination and MMPs were extracted from the other half for gelatin zymography. RESULTS: Marimastat inhibited neointimal thickening in a concentration-dependent manner; inhibition was significant at 10(-5) and 10(-6) mol/l (P=0.006). This observation was paralleled by a significant reduction in the levels of MMP-2 and MMP-9 in the tissues. CONCLUSION: Marimastat significantly reduced neointimal thickening in this laboratory model. MMP inhibitors may offer a potential therapeutic strategy in the prevention of intimal hyperplasia.

Endothelin-1 is a mediator of intimal hyperplasia in organ culture of human saphenous vein.

BACKGROUND: Endothelin-1 (ET-1) is a powerful vasoconstrictor and a potent mitogen for vascular smooth muscle cells. Excessive smooth muscle cell proliferation is a feature of intimal hyperplasia, the pathological lesion of vein graft stenosis. This study investigates the role of ET-1 in isolated human saphenous vein smooth muscle cells and also in an organ culture of the human saphenous vein. METHODS: Growth-arrested human saphenous vein smooth muscle cells were stimulated with ET-1 and proliferation quantified by [3H]thymidine uptake in these cells compared with unstimulated control cells. Organ cultures of the human saphenous vein were established with endothelium intact, with endothelium denuded, and with endothelium denuded and the culture medium supplemented with ET-1. RESULTS: ET-1 stimulated DNA synthesis in isolated smooth muscle cells in a dose-dependent manner with half-maximal stimulation at 1 nmol/l. Addition of ET-1 to denuded vein caused a significant increase in median (range) neointimal thickness, from 4 (0-19) to 20 (4-30) microns (P < 0.05). In veins cultured with ET-1 a parallel increase in median (range) neointimal proliferation index, from 21 (0-31) to 33 (23-46) per cent (P < 0.05), was also observed. CONCLUSION: These results demonstrate that ET-1 is a mediator of intimal hyperplasia in human saphenous vein in vitro. Endothelin receptor antagonists may therefore be of therapeutic value in the modulation of vein graft intimal hyperplasia.

Human saphenous vein organ culture: a useful model of intimal hyperplasia?

OBJECTIVES: Although cell culture techniques and animal models of intimal hyperplasia have increased our current understanding of the aetiology of vein graft stenosis, the results of such studies have been difficult to relate to the human situation. DESIGN: The present study was designed to validate an organ culture of human saphenous vein by comparing the changes occurring in cultured vein with those seen in pathological vein graft stenoses and to identify a suitable marker of cell proliferation. MATERIALS AND METHODS: Saphenous vein segments were cultured for 14 days, fixed in formalin and processed for immunohistochemistry. Freshly excised stenoses were fixed and processed similarly. A number of markers of cell proliferation were evaluated in the culture system in order to identify the one best suited to this particular model. RESULTS: Marked similarities were observed in the cellular and extracellular matrix composition, and electron microscopy revealed that both the neointima of the cultured vein and the pathological lesion contained an abundance of smooth muscle cells of a secretory phenotype. Bromodeoxyuridine proved to be the most reliable proliferation marker and revealed that early proliferation in the superficial layers of the vein intima gave rise to the formation of neointima. Both proliferation and neointimal thickness were maximal by day 14 in culture. Proliferation declined rapidly thereafter, and the neointima was maintained. CONCLUSIONS: The changes occurring in cultured vein and graft stenoses bore many similarities, thereby justifying the use of organ culture as a valuable experimental tool.

Osteopontin is not a marker for proliferating human vascular smooth muscle cells.

Osteopontin (OP) is a secreted glycoprotein that contains the Arg-Gly-Asp (RGD) cell-binding sequence that binds calcium and is chemotactic and adhesive for rat vascular smooth muscle cells (VSMCs). OP gene expression is upregulated in cultured rat VSMCs in vitro and after balloon carotid injury in vivo, suggesting that OP may be a marker for proliferating VSMCs in vivo and in vitro. Our in situ hybridization studies of human atherosclerotic coronary vessels, however, have shown OP mRNA expression in plaque macrophages but not VSMCs. The current study investigated OP mRNA expression in cultured human VSMCs and macrophages and in an organ culture model of neointima formation in human saphenous vein. OP mRNA expression was not detected by Northern blot analysis of total RNA from subconfluent or confluent cultures of human VSMCs of any passage maintained in normal growth medium or after stimulation with TGF beta 1 (20 ng/mL), angiotensin II (1 mumol/L), or basic fibroblast growth factor (10 mg/mL) but was just detectable after stimulation with activation vitamin D3 (1 mumol/L). In contrast, cultured human macrophages expressed high levels of OP mRNA that were not dependent on lipid loading. OP mRNA was detected in isolated foci in all layers of saphenous veins maintained in organ culture for 14 days, including <2% of neointimal cells, a distribution that paralleled that of tissue macrophages. These results suggest that OP gene expression is not a marker for proliferation of human VSMCs in vitro and highlight a fundamental difference in the biology of human and rodent VSMCs.