Role of redox signaling and poly (adenosine diphosphate-ribose) polymerase activation in vascular smooth muscle cell growth inhibition by nitric oxide and peroxynitrite

PURPOSE: The vascular mediator, nitric oxide regulates vascular smooth muscle cell proliferation and can react with superoxide to form peroxynitrite, a highly reactive free radical. The intracellular mechanisms by which nitric oxide and peroxynitrite inhibit smooth muscle cell growth remain undefined, as is the potential role of peroxynitrite formation in the antiproliferative effects of nitric oxide. We sought to define the intracellular effects and signaling mechanisms of nitric oxide and peroxynitrite in smooth muscle cells. METHODS: Cultured rat aortic smooth muscle cells were treated with exogenous nitric oxide or peroxynitrite and inhibitors of nitric oxide and redox signaling pathways. Cell growth, DNA synthesis, apoptosis, cyclic guanosine 3'-5' monophosphate (cGMP) levels, poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity, and cytotoxicity were assayed. Peroxynitrite formation was determined by nitrotyrosine immunoblotting. Vasoreactivity was assessed in isolated rat aortic rings after treatment with nitric oxide/peroxynitrite and redox agents. RESULTS: Both exogenous nitric oxide and peroxynitrite decreased cell growth and DNA synthesis of cultured rat aortic smooth muscle cells, but peroxynitrite-induced growth arrest was irreversible and associated with apoptosis and cytotoxicity. Inhibition of guanylate cyclase, PARP activity, mitogen-activated protein kinase, or bypass of ornithine decarboxylase did not reverse growth arrest by nitric oxide. The antioxidants N-acetylcysteine, ascorbate, and glutathione selectively reversed growth inhibition by nitric oxide but not by peroxynitrite. Antioxidants did not impair nitric oxide-induced cGMP generation in smooth muscle cells or nitric oxide-induced vasodilatation of isolated aortic rings. Nitric oxide treatment did not result in peroxynitrite formation and augmentation of superoxide levels did not induce peroxynitrite-like effects. Peroxynitrite-induced cytotoxicity and apoptosis were not reversed by antioxidants or PARP inhibition, because peroxynitrite activated PARP in J774 macrophages but failed to activate PARP in smooth muscle cells. CONCLUSIONS: Exogenous nitric oxide induces reversible cytostasis in smooth muscle cells by a redox-sensitive mechanism independent of peroxynitrite formation and distinct from the nitric oxide vasodilating mechanism. Peroxynitrite does not activate PARP selectively in smooth muscle cells and induces redox-independent smooth muscle cell cytotoxicity and apoptosis. Thus, the antiproliferative effects of nitric oxide and peroxynitrite on smooth muscle cells use divergent intracellular pathways with distinct redox sensitivities. These findings are relevant to the pathogenesis of vascular disease and the potential application of nitric oxide-based therapy for vascular disease. CLINICAL RELEVANCE: Vascular smooth muscle cell proliferation is an important component of atherosclerosis, vein graft failure, and arterial restenosis, and is known to be regulated by the vascular signaling molecule nitric oxide. Nitric oxide can combine with the free radical superoxide to form the unstable metabolite peroxynitrite, which has been detected in human vascular lesions. This study examines the role of peroxynitrite in mediating the antiproliferative effects of nitric oxide. We identify important differences in the effects and intracellular mechanisms of nitric oxide and peroxynitrite in regulating vascular smooth muscle cell proliferation and programmed cell death. Defining the differential effects of these free radicals in vascular cells is important to our understanding of the pathogenesis of vascular disease and the development of novel therapy aimed at treating proliferative vascular lesions.     

Cell biology of human vascular smooth muscle.

Vascular smooth muscle is the cellular substrate of most significant arterial diseases. Restenosis after angioplasty and surgery mainly represents vascular smooth muscle reaction to trauma, a process which is also significant in the early stages of atherogenesis. Empirical approaches, based on findings in animal models of vascular injury, have notably failed to make any impact on human restenosis. We have developed and validated growth of the human VSMC in culture as a model of restenosis. Intimal hyperplastic lesions producing vascular restenosis contain cells that have reduced sensitivity to physiological growth inhibition by heparin in cell culture conditions, compared with cells from normal vascular tissue. Undiseased saphenous vein obtained from patients with intimal hyperplastic restenoses also contain cells that are relatively resistant to heparin inhibition. Arterial healing that progresses to restenosis may have distinct and fundamental differences at the cellular level from the normal process of arterial healing after injury.     

An IkappaB-alpha mutant inhibits cytokine gene expression and proliferation in human vascular smooth muscle cells.

BACKGROUND: Inflammatory reaction and intimal proliferation of smooth muscle cells are characteristics of vascular stenotic lesions. Nuclear factor kappaB (NF-kappaB) is involved in regulation of inflammation and cell survival in a variety of cell types. We tested a hypothesis that selective inhibition of NF-kappaB by expression of a mutated, nondegradable inhibitor of NF-kappaB, IkappaB-alphaM, would inhibit proinflammatory cytokine expression and proliferation in human vascular smooth muscle cell. MATERIALS AND METHODS: Smooth muscle cells were cultured from internal mammary artery and infected with recombinant adenovirus vectors. RESULTS: Adenoviral expression of IkappaB-alphaM inhibited diverse signal-triggered cellular IkappaB-alpha degradation, subsequent NF-kappaB activation, and transactivation of proinflammatory cytokine genes. Expression of IkappaB-alphaM in low-density VSMC led to a 60% reduction in serum-stimulated cell growth and a 10% increment in apoptotic incidence but was without effect in high-density cultures. Coexpression of NF-kappaB p65 attenuated apoptosis in low-density cells induced by IkappaB-alphaM. Therefore, the susceptibility to apoptosis induction in the low-density cells correlated with lower constitutive NF-kappaB activity. The induction of apoptosis by IkappaB-alphaM and the rescue by NF-kappaB p65 might be explained by mutual control of NF-kappaB p65 and IkappaB-alphaM access to the nucleus. CONCLUSION: Our results suggest that expression of nondegradable IkappaB-alpha might have therapeutic potential in both vascular inflammatory reaction and smooth muscle cell proliferation.     

细胞凋亡和血管病变

目的 探讨细胞凋亡与血管病变的关系。方法 就细胞凋亡的概念、特征、基因调控、信号传导及其与血管病变的关系作一综述。结果 细胞因子、辐射、氧自由基等可诱导血管内皮细胞凋亡、导致血管损伤;而平滑肌细胞凋亡与动脉粥样硬化和血管成形术后再狭窄密切相关。结论 细胞凋亡异常与血管病变密切相关。     

核因子-κB与血管成形术后再狭窄

目的探讨核因子-kappaB(NF—κB)在血管成形术后再狭窄中的作用。方法对国内、外近5年的有关文献进行综述。结果在血管成形术后NF-κB可引起平滑肌细胞增生及凋亡减少，导致血管内膜过度增生。结论NF—κB在血管成形术后再狭窄中起着重要作用。     

Mutual exclusion of apoptosis and hsp70 in human vein intimal hyperplasia in vitro

BACKGROUND: Apoptosis of vascular smooth muscle cells (VSMC) plays a role in physiological vascular remodeling, as well as in disease states such as atherosclerosis and restenosis after angioplasty. Heat shock protein 70 (hsp70) may protect the cell against apoptosis and/or necrosis. In this study, we examined hsp70 expression and its temporal relationship to cell survival or death in a model of intimal vein hyperplasia in vitro. METHODS: Segments of human saphenous veins were placed into culture. At different days vein segments were serum-starved or exposed to heat shock. Apoptosis and hsp70 expression were analyzed by Western blot, immunohistochemistry, and TUNEL assay. RESULTS: A marked intimal vein hyperplasia developed after 14 days of culture when compared with baseline. hsp70 was present at baseline and disappeared during culture. Heating during culture could not up-regulate hsp70. The apoptotic markers were absent at baseline and present during normal culture. Conversely, serum starvation stimulated strong hsp70 expression coincidental with the disappearance of apoptotic markers. CONCLUSIONS: Stimulation of veins during culture with serum resulted in hyperplasia, apoptosis, and inhibition of hsp70 expression. Down-regulation of hsp70 may permit apoptosis and vessel wall remodeling in this model.     

Concomitant blockade of platelet-derived growth factor receptors alpha and beta induces intimal atrophy in baboon PTFE grafts

OBJECTIVE: Although current treatments for restenosis attempt to prevent the development of intimal hyperplasia, an alternative strategy is to induce intimal atrophy after restenosis has developed. Because platelet-derived growth factor (PDGF) is a smooth muscle cell growth and survival factor, we tested the hypothesis that complete blockade of PDGF by using antibodies against PDGF receptors alpha and beta would cause intimal atrophy in a baboon vascular graft model. METHODS: We administered chimeric antibodies against PDGF receptor alpha or PDGF receptor beta, either separately or together, to baboons with bilateral prosthetic aortoiliac grafts, the intimas of which had reached maximal size before treatment was begun. High blood flow, which we have previously shown to cause intimal atrophy, was induced through one graft to serve as a positive control. After 2 weeks, the intima lining the grafts was assessed for cross-sectional area, cell proliferation, and apoptosis by standard morphologic and immunohistochemical techniques. RESULTS: Blocking both PDGF receptors simultaneously reduced the cross-sectional area of the normal-flow graft intima by 44% (P <.05 vs control), whereas treatment with the individual antibodies did not significantly alter intimal area. Blockade of both receptors also inhibited smooth muscle cell proliferation by 66% (P <.05 vs control), whereas neither antibody alone altered proliferation. In contrast, all treatments increased smooth muscle cell apoptosis threefold to fivefold. CONCLUSIONS: These data suggest that simultaneous inhibition of cell proliferation and stimulation of cell death by the administration of antibodies to both PDGF receptor alpha and receptor beta is required for intimal atrophy in this baboon graft model. In addition, these data provide an in vivo model for the pharmacologic induction of intimal atrophy and introduce a novel clinical approach to treat intimal hyperplasia.Clinical relevance This study introduces the concept of pharmacologic induction of intimal atrophy. Intimal hyperplasia plagues all forms of arterial reconstruction. Currently, the only effective treatment of these restenotic lesions is balloon angioplasty or operative revision. An alternative approach to patients with clinically significant intimal hyperplasia might be to stimulate intimal regression by modulating growth and survival factors required for intimal maintenance. Although PDGF is known to be critical in intimal formation, the results of this study suggest that PDGF is also critical for intimal maintenance. Inhibition of the PDGF system may prove to be a clinically applicable approach for inducing intimal atrophy.     

EGF receptor activity modulates apoptosis induced by inhibition of the proteasome of vascular smooth muscle cells

The observation that intracellular protein turnover rates participate directly in cell viability led to the development and clinical use of potent proteasome inhibitors. This study determined that the mechanism of apoptosis that is induced by inhibition of the proteasome of vascular smooth muscle cells (VSMC) was related to the intracellular accumulation of Bad, a BH3-only member of the Bcl-2 family of apoptosis regulators. Experiments confirmed that the apoptotic process was mitochondria- and caspase-dependent. Ubiquitination and accumulation of Bad in VSMC followed inhibition of the proteasome, and depletion of Bad using RNA interference prevented apoptosis that was induced by proteasome inhibition with PS-341. EGF receptor (EGFR) activation produced posttranslational modifications of Bad, providing the pro-survival signals that prevented apoptosis of smooth muscle cells during proteasome inhibition. Antagonists of the EGFR potentiated the apoptotic rate. In summary, the activities of the EGFR and the proteasome focused on Bad and the intrinsic apoptotic pathway and were involved integrally in determining viability of VSMC. These findings might prove useful in the management of diseases in which proliferation of vascular smooth muscle cells plays a central role.     

靶向STAT3的慢病毒表达载体的构建及其对血管平滑肌细胞增殖凋亡的影响

目的 构建大鼠STAT3基因的shRNA慢病毒表达载体,并观察其对大鼠血管平滑肌细胞增殖和凋亡的影响.方法 针对STAT3基因的不同部位设计4对shRNA的寡核苷酸片段,克隆到慢病毒载体PLKO.1中,构建靶向STAT3基因的慢病毒载体PLK0.1-STAT3-shRNA,检测并筛选最佳抑制效率的shRNA干扰载体.并将其转染大鼠血管平滑肌细胞,用噻唑蓝法和流式细胞仪检测沉默STAT3基因后对血管平滑肌细胞增殖和凋亡能力的影响.结果　靶向STAT3慢病毒表达载体构建成功.转染PLKO.1-STAT3-shRNA后,STAT3蛋白表达明显下降,其中以PLKO.1-STAT3-S1最为明显,达到90%以上;转染PLKO.1-STAT3-S1的细胞增殖能力(A值=0.25±0.05)明显低于未转染组(A值=0.62±0.12)和阴性对照组细胞(A值=0.59±0.11)(P＜0.05);而早期细胞凋亡率(26.9±2.8)%和晚期细胞凋亡率(9.5±1.6)%均明显高于未转染组和阴性对照组(P＜0.01).结论　成功构建并筛选最佳抑制效率的靶向STAT3慢病毒表达载体PLKO.1-STAT3-S1,该载体能有效抑制大鼠血管平滑肌细胞增殖,并促进细胞凋亡.

Abstract：

Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector carrying STAT3 gene in rats, and to investigate its effects on proliferation and apoptosis of vascular smooth muscle cells by silencing STAT3. Methods Four oligonucleotides targeting STAT3 gene were synthesized and cloned into lentivirus vector PLKO. 1. The shRNA lentiviral vector with best transfection efficiency was detected and identified, which was transfected into vascular smooth muscle cells in rats, and its effects on proliferation and apoptosis of vascular smooth muscle cells were measured by MTT and flow cytometry after silencing STAT3. Results The recombinant lentivirus vector PLKO. 1-STAT3-shRNA was constructed successfully. PLKO. 1-STAT3-shRNA knocked down the expression of STAT3 protein dramatically, especially PLKO. 1-STAT3-S1, whose transfection efficiency was more than 90%. The proliferation capacity of vascular smooth muscle cells transfected with PLKO. 1-STAT3-S1 (A value =0. 25 ±0. 05 ) was significantly lower than no-transfected group (A value =0. 62 ±0. 12) and negative control group (A value =0. 59 ±0. 11 )(P ＜ 0. 05). Meantime the early apoptosis rate (26. 9 ± 2. 8 ) % and late apoptosis rate (9. 5 ± 1.6 ) % in PLKO. 1-STAT3-shRNA-transfected group were significantly higher than in no-transfected group and negative control group (P ＜ 0. 01 ). Conclusion The recombinant lentivirus shRNA vector targeting STAT3,PLKO. 1-STAT3-S1, with best transfection efficiency, is constructed successfully. PLKO. 1-STAT3-S1 can inhibit the proliferation of vascular smooth muscle cells, and promote the cell apoptosis. This study lays the foundation for further studying on targeting treatment of vascular restenosis.     

Vascular and other non-tumour applications of photodynamic therapy

Photodynamic therapy is being investigated as a cancer therapy. As a cytotoxic treatment, it may also have therapeutic benefits in certain non-tumour conditions. The mechanism of photodynamic therapy is discussed in relation to its cancer therapy. The literature on non-tumour applications of photodynamic therapy is subsequently reviewed, highlighting its vascular applications in particular.Arterial angioplasty restenosis has proved resistant to all treatments tried thus far. Because fibrocellular intimal hyperplasia arising from the proliferation of vascular medial smooth muscle cells forms the pathological basis of restenosis, photodynamic therapy has been considered in its prevention. The literature on two second-generation photosensitizers (5-aminolaevulinic acid and phthalocyanine) which are likely to achieve clinical application are reviewed with regard to their photodynamic effects on fibrocellular intimal hyperplasia.This review concludes that photodynamic therapy shows enough promise for the inhibition of fibrocellular intimal hyperplasia for large animal studies to be pursued.     

Nicotine inhibits apoptosis and stimulates proliferation in aortic smooth muscle cells through a functional nicotinic acetylcholine receptor

Atherosclerosis and neointimal hyperplasia formation are induced by alterations in the homeostatic balance between cell growth and cell death. Apoptosis is a physiological cell death process that, when deregulated, may be involved in many pathological conditions. Cigarette smoking is a primary risk factor for vascular disease and nicotine seems to exert its atherogenic effects in part through the increase of smooth muscle cell (SMC) proliferation. The aim of this study was to investigate the effect of nicotine on SMC apoptosis. Nicotine added for 24 and 72 h to serum deprived cell cultures resulted in a decrease of apoptotic SMCs. The inhibition was direct and not mediated by platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor beta(1), autocrinally released by nicotine-treated SMCs, because it was not influenced by addition of specific neutralizing antibodies. Apoptosis inhibition as well as the proliferation increase, and basic fibroblast growth factor expression on nicotine-treated SMCs were blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha subunits of nicotinic receptor. In conclusion, we propose that nicotine could lead to the increase of neointimal SMCs in vascular lesions by inducing the inhibition of physiological SMC apoptosis and the increase of SMC proliferation. We also showed that nicotine signaling occurs as a result of activation of the classical nicotine receptor pathways.     

Nitric oxide: implications for vascular and endovascular surgery.

Nitric oxide has a key role in vascular homeostasis. It plays a protective role by suppressing abnormal proliferation of vascular smooth muscle following various pathological situations including atherosclerosis and restenosis after vascular interventions such as balloon angioplasty, stent deployment and bypass grafting. It also has strong antiplatelet and anti-thrombogenic properties. In this review, possible applications to daily vascular and endovascular surgery practice, including systemic use of NO donors, enhancing endogenous production of NO by L-arginine and gene therapy, local delivery strategies and coating stents and grafts with NO-delivering/enhancing chemicals are reviewed.     

自体移植静脉再狭窄基因水平的研究进展

为防治移植静脉再狭窄,基因水平的研究显然具有重要意义.笔者就近年来国内外针对移植静脉再狭窄的基因水平的探索进行综述,主要内容包括应用促血管再内皮化基因、抗血栓形成基因、抑制血管平滑肌细胞增殖和胞外基质沉积基因以及联合多基因共同治疗移植静脉再狭窄的研究,并指出未来基因水平研究发展方向.     

Sodium-dependent phosphate cotransporters and vascular calcification

PURPOSE OF REVIEW: Vascular calcification is associated with cardiovascular events in patients with end-stage renal disease and diabetes. Hyperphosphatemia is a risk factor for vascular calcification in these patients. Sodium-dependent phosphate cotransporters are required for cellular phosphate uptake. This review focuses on the potential role of phosphate transport and type III sodium-dependent phosphate cotransporters in the process of vascular calcification. RECENT FINDINGS: Consistent with clinical and animal studies, elevated phosphate induces mineralization of cultured smooth muscle cells in vitro. Calcification is concomitant with osteochondrogenic phenotype change in smooth muscle cells characterized by induction of osteochondrogenic differentiation marker, Runx2, and inhibition of smooth muscle cell lineage marker, SM22. Inhibition of the type III sodium-dependent phosphate cotransporter, Pit-1, blocks phosphate-induced smooth muscle cell calcification. Moreover, the phosphate-induced osteochondrogenic phenotype modulation is also abrogated by Pit-1 inhibition. Pit-1 is upregulated by several calcification-promoting factors, including tumor necrosis factor-alpha, bone morphogenetic protein 2, platelet-derived growth factor and elevated calcium. SUMMARY: Phosphate uptake via Pit-1 is required for osteochondrogenic phenotypic change and calcification of vascular smooth muscle cells in vitro. Modulation of Pit-1 expression or its transport activity may provide a novel therapeutic target for intervention of vascular calcification.     

Photodynamic therapy in cardiac and vascular surgery

One of the major drawbacks to therapeutic cardiovascular intervention is intimal hyperplasia and constrictive remodeling, which result in vascular restenosis. Neointimal hyperplasia is characterized by proliferation and migration of smooth muscle cells. These cells also produce new extracellular matrix, leading to narrowing of vessels. Photodynamic therapy (PDT) represents one of the most promising approaches to the inhibition of intimal hyperplasia. PDT requires the interaction among 3 factors: a source of light, usually a laser, a photosensitizer and oxygen. When the inert photosensitizer absorbs light of a specific wavelength, it is activated to an excited triplet state, generating reactive oxygen species. These free radicals are able to induce apoptosis of the smooth muscle cells that had absorbed the photosensitizer; they also induce changes in the extracellular matrix, reducing cell migration. Because of continued success of PDT in inhibiting intimal hyperplasia in experimental animal models, it is now being tested in clinical trials for vascular diseases. PDT offers many advantages to the surgeon since it can act on numerous factors responsible for vascular lesions. In the future PDT could be used in helping to overcome the inherent failures associated to vascular reconstruction. This treatment modality is emerging as an encouraging therapeutic option, either alone or as an adjunct to conventional treatment. However, more detailed clinical investigation are necessary to determine its full potential.     

Ambient pressure upregulates nitric oxide synthase in a phosphorylated-extracellular regulated kinase- and protein kinase C-dependent manner

PURPOSE: Using endothelial cell/smooth muscle cell (SMC) cocultures, we have demonstrated that pressurized endothelial cell coculture inhibits SMC proliferation and promotes apoptosis, and that this effect is transferable through pressurized endothelial medium. We now hypothesized that endothelial nitric oxide synthase (eNOS) plays a significant role in mediating these pressure-induced effects. METHODS: Conditioned media from endothelial cells and SMCs exposed to ambient and increased pressure were transferred to recipient SMCs. We counted cells after 5 days of incubation with these media and evaluated eNOS and inducible NOS (iNOS) levels by Western blot. RESULTS: Conditioned media from pressurized endothelial cells significantly decreased recipient SMC counts. This effect was sustained when N-nitro-L-arginine-methyl ester (L-NAME) was added to recipient cells but abolished when L-NAME was added to donor cells. SMCs were then exposed to control and pressurized conditions in monoculture or in coculture with endothelial cells. Pressure and coculture caused similar increase in iNOS levels but had no additive effect in combination. Finally, endothelial cells were exposed to control and pressurized environments. Pressure caused a 24% +/- 1.6% increase in eNOS protein (P = .04, n = 12). This effect was sustained when cells were treated with L-NAME (32% +/- 1.6% increase, P = .02) but abolished when endothelial cells were treated with calphostin C or PD98059 to block protein kinase C (PKC) or extracellular regulated kinase (ERK). Pressure also increased endothelial phosphorylated ERK (p-ERK) by 1.8-fold to 2.6-fold compared with control conditions after exposure of 2, 4, and 6 hours (P = .02, n = 4). This increase was sustained after pretreatment with calphostin C. CONCLUSION: Pressure modulates endothelial cell effects on SMC growth by increasing eNOS in an ERK-dependent and PKC-dependent manner. CLINICAL RELEVANCE: Intimal hyperplasia is the main cause for restenosis that complicates 10% to 30% of all such vascular procedures and 30% to 40% of endovascular procedures. This article provides some novel information about smooth muscle cell/endothelial cell interaction, one of the main regulators of vascular remodeling and intimal hyperplasia. The role of endothelial cell/smooth muscle cell interaction cannot be studied well in vivo because these interactions cannot be distinguished from other factors that coexist in vivo, such as flow dynamics, matrix proteins, inflammatory factors, and interactions with other cells in the vascular wall and in the bloodstream. In this work, we use pressure as a triggering stimulus to alter in vitro endothelial behavior and identify important changes in endothelial regulation of smooth muscle cell biology. The pathways involved in this process and discussed in this article could ultimately be used to manipulate endothelial cell/smooth muscle cell interaction in clinical disease.     

Platelet-derived growth factor and its role in atherogenesis: a brief review.

A number of locally acting growth factors are now widely understood to be important in the regulation of cellular activity. Platelet-derived growth factor (PDGF) is one of the best known and is an important mitogenic stimulant of vascular tissues. PDGF is stored primarily by platelets but other cells, including endothelium and smooth muscle, can act as sources. The mitogenic properties of PDGF may be influential in the pathogenesis of atherosclerosis. PDGF has access to the vascular intima and may act upon smooth muscle cells to produce proliferation, a primary feature of atherosclerosis. In this brief review we examine the structure, function and influences of PDGF with special emphasis on its potential role in the pathogenesis of atherosclerosis.     

PLATELET-DERIVED GROWTH FACTOR AND ITS ROLE IN ATHEROGENESIS: A BRIEF REVIEW

A number of locally acting growth factors are now widely understood to be important in the regulation of cellular activity. Platelet-derived growth factor (PDGF) is one of the best known and is an important mitogenic stimulant of vascular tissues. PDGF is stored primarily by platelets but other cells, including endothelium and smooth muscle, can act as sources. The mitogenic properties of PDGF may be influential in the pathogenesis of atherosclerosis. PDGF has access to the vascular intima and may act upon smooth muscle cells to produce proliferation, a primary feature of atherosclerosis. In this brief review we examine the structure, function and influences of PDGF with special emphasis on its potential role in the pathogenesis of atherosclerosis.     

The effect of von hippel-lindau gene transfer on human vascular smooth muscle cell proliferation and apoptosis

Von Hippel-Lindau (VHL) gene is a tumor suppressor gene that plays a genome "gatekeeper' role and controls several downstream effector genes. We have previously demonstrated that both in vivo and in vitro adenovirus-mediated gene transfer of tumor suppressor genes into the vascular endothelium is effective in decreasing neointimal hyperplasia and abnormal cell proliferation. The degree of apoptosis induced by these genes is critical in mediating the in vivo responses to gene therapy and the maintenance of the crucial balance between cell death and viability. Since VHL gene is known to regulate vascular endothelial growth factor (VEGF) as well as other angiogenic factors, it may exhibit a greater potential in the attenuation of vascular disorders in comparison to other tumor suppressor genes. This study focused on whether adenovirus-mediated VHL gene transfer into human vascular smooth muscle cells has an effect on cell proliferation and induction of apoptosis. Human aortic smooth muscle cells (HASMC) were grown as monolayers and transfected with varying titers of adenovirus containing the VHL cDNA (AdVHL). The negative controls were adenovirus containing green fluorescent protein (AdGFP), vector alone (AdNull), and virus-free infection medium. Adenovirus encoding wild-type p53 (Adp53) was used as positive control. Cell viability and proliferation were determined by using trypan blue exclusion and MTS-based CellTiter 96 AQ Proliferation Assay. Apoptosis was evaluated by TUNEL assay, morphologic changes, and nucleosomal DNA degradation. Following AdVHL transfection HASMCs demonstrated a dose-dependent decrease in viability as compared to negative controls (p < 0.05). AdVHL-transfected cells exhibited a decrease in their proliferative ability by 40.21 +/-1.66 (SEM)%. In cultures transfected with the positive control, Adp53, the cell viability as well as proliferation was highly reduced (p < 0.001). AdGFP and AdNull did not increase HASMC apoptosis above baseline levels. The cells exposed to adenoviruses expressing tumor suppressor genes underwent apoptosis, with Adp53 demonstrating a very high magnitude of cell death (75.27 +/-3.52 [SEM]%). AdVHL expression caused 45.36 +/-2.55 (SEM)% apoptosis in HASMC. Recombinant adenovirus-mediated VHL expression is efficacious in limiting vascular smooth muscle cell growth in vitro. Overexpression of VHL suppresses HASMC proliferation and regulates apoptosis. Further experiments are indicated to examine whether VHL may be a useful adjunct in limiting myointimal hyperplasia.