Hyperlipemic-very low density lipoprotein, intermediate density lipoprotein and low density lipoprotein act synergistically with serotonin on vascular smooth muscle cell proliferation

BACKGROUND: Previous studies have shown that very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) from hyperlipidemic plasma are more atherogenic than those from normal plasma. Since platelet aggregation at sites of atherosclerotic injury exposes the cells to high concentrations of serotonin (5HT), a known mitogen for vascular smooth muscle cells (VSMCs), it was examined whether VLDL, IDL or LDL from plasma of 1% cholesterol-fed rabbits can potentiate the mitogenic effect of 5HT on VSMC. METHODS: Growth arrested primary aortic VSMC in 1st or 2nd passage were incubated with different concentrations of VLDL, IDL or LDL in the presence or absence of pertusis toxin (PTX) for 24 h followed by incubation with 5HT for 24 h. The amount of [3H]thymidine incorporated into the DNA as well as the increase in cell number was measured. RESULTS: Either VLDL, IDL or LDL at a concentration of 60 microg/ml induced proliferation of VSMC by themselves (196, 137 or 122% increase in [3H]thymidine incorporation, or 122, 119 or 122% increase in cell number, respectively when compared to the control, P<0.05). This effect on DNA synthesis was markedly potentiated by 50 microM 5HT to 465, 714 and 1369%, respectively. PTX reversed the mitogenic effect of 5HT, but not that of VLDL, IDL or LDL. Conclusion: These results suggest that even low concentration of VLDL, IDL or LDL from hypercholesterolemic plasma may significantly potentiate the mitogenic effect of 5HT, that is released by aggregating platelets at sites of vascular damage.     

氧化低密度脂蛋白损伤血管内皮细胞与平滑肌细胞增殖相关性研究

氧化修饰低密度脂蛋白(oxidized low-densitylipoprotein,ox-LDL)损伤血管内皮细胞(VEC)是动脉粥样硬化(AS)形成的重要因素之一。VEC结构和功能的损伤及血管平滑肌细胞(VSMC)增殖和迁移是AS形成及发展过程中重要的病理生理改变。流行病学资料表明,血脂水平高于正常与AS的发生及冠心病(CHD)的发病率有密切的相关性,从而明确高脂血症是AS及CHD的危险因素。许多学者认为,VEC结构和功能损伤是AS发生的始动环节,VSMC增殖和迁移是AS发生及发展的关键病理环节。人们在预防和治疗AS过程中,不仅要着眼于改善低密度脂蛋白(LDL)的氧…     

Effect of glycated low density lipoprotein on smooth muscle cell proliferation.

BACKGROUND: We investigated the effect of glycated low density lipoprotein (LDL) on smooth muscle cell proliferation. METHODS: Blood was drawn from 6 healthy subjects after overnight fasting. Native LDL was obtained by separating LDL from the samples with sequential ultracentrifugation. Glycated LDL was prepared by glycating the native LDL in vitro. Native and glycated LDL were added to a medium containing cultured porcine coronary artery smooth muscle cells, and the change in cell proliferation was examined after 24, 48, 72, and 96 hs. The cells were counted using a cell counting kit (Dojin Chemical Co., Ltd.). RESULTS: There was no significant difference in the cell count between the control group, in which only PBS was added, and the native LDL group. However, cell proliferation was appreciably higher in the glycated LDL group than in the native LDL group. The mean total cell count at 24, 48, 72 and 96 hs was significantly higher (p<0.01) in the glycated LDL group (median: 0.843; range: 0.576-1.060) than in the native LDL group (median: 0.541; range: 0.282-0.683). CONCLUSIONS: These findings suggest that glycated LDL induces significantly greater acceleration of smooth muscle cell proliferation than does native LDL. Therefore, the acceleration of smooth muscle cell proliferation requires modification of LDL.     

大黄素自身抑制血管平滑肌细胞迁移和增殖

目的探讨大黄素对血管平滑肌细胞迁移和增殖的作用以及大黄素在血管平滑肌细胞中是否存在代谢过程。方法采用Transwell迁移系统和MTT法观察大黄素对血管平滑肌细胞迁移和增殖的影响。结果大黄素能显著抑制平滑肌细胞迁移,5μg/mL大黄素抑制率为83·3%;大黄素呈浓度和时间依赖性抑制平滑肌细胞增殖。然而,血管平滑肌细胞作用24h后,上清液中的大黄素浓度并无显著降低;细胞色素p450氧化酶诱导剂或者抑制剂没有改变大黄素的细胞毒性作用或者细胞内活性氧水平。大黄素的主要代谢酶(细胞色素p450氧化酶)基因表达水平没有显著的上调。结论大黄素能够抑制血管平滑肌细胞的迁移和增殖,并且不被平滑肌细胞代谢,可能作为药物涂层支架的药物。     

Synthetic alphaVbeta3 antagonists inhibit insulin-like growth factor-I-stimulated smooth muscle cell migration and replication.

Porcine aortic smooth cells respond to insulin-like growth factor-I (IGF-I) with increases in DNA synthesis and cell migration. Because ligand occupancy of the alphaVbeta3 integrin has been shown to be necessary for IGF-I to stimulate maximal increases in both processes, we determined whether synthetic alphaVbeta3 antagonists could inhibit IGF-I-stimulated actions on this cell type. Low-molecular-weight compounds that had been selected based on their ability to compete with vitronectin for binding to purified human alphaVbeta3 in vitro were analyzed for their ability to compete with 125I-kistrin (a known ligand for porcine alphaVbeta3) for binding to porcine alphaVbeta3. Nine compounds were screened, and five were found to be potent competitive inhibitors. The most potent compound, SC-69000, resulted in 88% competition at 10(-7) M and was nearly equipotent with echistatin. The compounds that were the most potent inhibitors of kistrin binding were tested for their capacity to inhibit the cell migration response to IGF-I. Three compounds caused between 81-88% inhibition of IGF-I-stimulated migration at 10(-7) M. To determine whether these compounds could inhibit other IGF-I-stimulated actions, their ability to inhibit IGF-I-stimulated [3H]-thymidine incorporation into DNA was analyzed. The four compounds that were the most potent inhibitors of cell migration also inhibited IGF-I-stimulated DNA replication. IGF-I stimulates the synthesis of IGF binding protein-5 by these cells. Preincubation with the four most active compounds also resulted in significant inhibition of the ability of IGF-I to stimulate IGF binding protein-5 synthesis. AlphaVbeta3 occupancy by the ligand vitronectin has been shown to enhance the capacity of IGF-I to activate its receptor tyrosine kinase. The four most active compounds were shown to inhibit IGF-I-stimulated IGF-I receptor autophosphorylation. These findings suggest that blockade of ligand occupancy of the alphaVbeta3 integrin globally inhibits several IGF-I-stimulated biologic actions and that synthetic inhibitors are very active in this regard. Because these compounds can be administered to whole animals, they should be very useful in determining whether blocking alphaVbeta3 occupancy in vivo results in alteration in responsiveness to IGF-I.     

Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.

Vascular smooth muscle cells (VSMCs) proliferate in response to arterial injury. Recent findings suggest that, in addition to platelet-derived growth factors, growth factors from inflammatory cells and endothelial cells at the site of injury may contribute to VSMC proliferation. We hypothesized that a common mechanism by which endothelial cells and inflammatory cells stimulate VSMC growth could be the active oxygen species (i.e., O2-, H2O2, and .OH) generated during arterial injury. Using xanthine/xanthine oxidase to generate active oxygen species, we studied the effects of these agents on VSMC growth. Xanthine/xanthine oxidase (100 microM xanthine and 5 microunits/ml xanthine oxidase) stimulated DNA synthesis in growth-arrested VSMCs by 180% over untreated cells. Administration of the scavenging enzymes superoxide dismutase and catalase demonstrated that H2O2 was primarily responsible for xanthine/xanthine oxidase-induced VSMC DNA synthesis. H2O2 directly increased VSMC DNA synthesis and cell number (maximal at 200 microM) but decreased DNA synthesis of endothelial cells and fibroblasts. This effect was protein kinase C independent: sphingosine, a potent protein kinase C inhibitor, failed to block H2O2-induced VSMC DNA synthesis. H2O2 (200 microM) stimulated c-myc and c-fos mRNA levels by fourfold and 20-fold, respectively, as compared with quiescent levels. In contrast to DNA synthesis, H2O2 induction of c-myc and c-fos mRNA was primarily protein kinase C dependent. These findings show that H2O2 specifically increases VSMC DNA synthesis and suggest a role for this oxidant in intimal proliferation, especially after arterial injury.     

Angiotensin II stimulates synthesis of vascular smooth muscle cell proteoglycans with enhanced low density lipoprotein binding properties

One mechanism by which Angiotensin II (AII) may promote atherogenesis is through modulation of proteoglycan (PG) metabolism by vascular smooth muscle cells (SMC). To test this hypothesis, we investigated the effect of AII on PG synthesis by human aortic SMC and the ability of the newly synthesized PG to bind low density lipoprotein (LDL). AII stimulated PG synthesis by SMC in a dose- and time-dependent manner. In the presence of 1 microM AII, medium and cellular PG increased by 73 and 97%, respectively. AII caused a 55% increase in biglycan mRNA which resulted in a 52% increase in biglycan synthesis. Losartan, an AII receptor antagonist, and broad and isoform-specific protein kinase C (PKC) inhibitors abolished the AII-induced up-regulation of PG synthesis. Moreover, direct activation of PKC with phorbol ester stimulated PG synthesis significantly. Similarly, inhibitors of tyrosine kinase also caused inhibition of PG synthesis. AII increased the size and charge density of the newly synthesized PG. In addition, AII stimulated the synthesis of PG that bound LDL with very high affinity by 2.5-fold to 3-fold over control. These results suggest that the AII-mediated alterations in vascular SMC PG metabolism may contribute to the pathophysiology of atherosclerosis.     

Mechanisms of hepatocyte growth factor-mediated vascular smooth muscle cell migration

The migration of vascular smooth muscle cells (SMCs) from the media into the neointima and their subsequent proliferation is important in the pathogenesis of atherosclerosis. This process is regulated by multiple factors, including growth factors, and involves changes in the interaction of SMCs with the extracellular matrix and in intracellular signaling cascades that regulate cell movement. We demonstrated previously that hepatocyte growth factor (HGF) is expressed in human atherosclerotic plaques. Although HGF has been shown to promote SMC migration, the mechanisms involved in this process have not been characterized fully. In this study, inhibitory antibodies were used to determine which integrins mediated HGF-induced SMC migration. Inhibition of beta1 or beta3 integrin resulted in a significant decrease in migration. Subsequent experiments were performed to characterize additional biochemical mechanisms involved in HGF-mediated migration. HGF induced the redistribution of focal adhesions, the activation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and their increased association with beta1 and beta3 integrins, and the production of pro-matrix metalloproteinase-2. Migration levels were significantly reduced by cotreatment of SMCs with the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, UO126, the p38 inhibitor, SB203580, or the phosphatidylinositol-3 kinase inhibitor, LY294002. In HGF-treated SMCs, focal adhesion redistribution and FAK and Pyk2 activation were decreased by ERK1/2 inhibition. Neither SB203580 nor LY294002 inhibited HGF-induced ERK1/2 activation. Thus, ERK1/2 signaling may play an important role in HGF-mediated SMC migration by contributing to focal adhesion redistribution and FAK and Pyk2 activation.     

Thromboxane and vascular smooth muscle cell growth in genetically hypertensive rats

The vascular wall has the capacity to produce thromboxane A2. However, the role of vascular thromboxane A2 is still uncertain. In this study, we examined the relationship between vascular thromboxane A2 generation and vascular smooth muscle cell growth in spontaneously hypertensive rats (SHR). Vascular thromboxane A2 generation was significantly enhanced by 49% in 5-week-old and by 117% in 15-week-old SHR as compared with age-matched Wistar-Kyoto rats (WKY). Thromboxane A2 generation was also significantly enhanced by 59% in the cultured vascular smooth muscle cells of SHR when compared with production in WKY. Vascular smooth muscle cells of SHR exhibited a significantly shortened doubling time (by 32%) and greater [3H]thymidine uptake (by 56%), as compared with those of WKY. OKY 046 (10(-5) M), a thromboxane synthase inhibitor, significantly tempered the rapid vascular smooth muscle cell growth in SHR by 9% for doubling time and by 10% for [3H]thymidine uptake. OKY 046 did not influence the doubling time of WKY. Conversely, a stable analogue of thromboxane A2 dose-dependently stimulated the [3H]thymidine uptake by vascular smooth muscle cells of WKY, and, at a concentration of 10(-5) M, shortened the doubling time of vascular smooth muscle cells of WKY by 11%, whereas it showed slight effects on SHR. These data indicate that vascular thromboxane A2 is involved in the regulatory mechanism of vascular smooth muscle cell growth and that enhanced vascular thromboxane A2 generation is partly responsible for the rapid proliferation of vascular smooth muscle cells of SHR. The alterations of vascular thromboxane production may be a key trait for genetic hypertension.     

Nerve growth factor synthesis in vascular smooth muscle.

Details of the interdependent, trophic relation between smooth muscle and its neural innervation are not well known despite suggestions that neural influences may contribute significantly to hypertensive and other cardiovascular disease. Vascular smooth muscle is a major target of innervation by neurons of the sympathetic nervous system. Sympathetic neurons depend on a constant supply of the potent neurotrophic peptide nerve growth factor. Nerve growth factor regulates an impressive list of neuronal and perhaps muscle properties, yet its source in vessels and the determinants of its synthesis are not known. We have taken advantage of the cytoarchitecture of the aorta to demonstrate that vascular smooth muscle cells synthesize nerve growth factor. The survival of cultured sympathetic neurons is supported in a nerve growth factor-dependent manner by co-culture with pure rat aortic vascular smooth muscle cells. Furthermore, pure smooth muscle cell cultures contain nerve growth factor-specific messenger RNA. Levels of messenger nucleic acid coding for nerve growth factor in smooth muscle are regulated by contractile agonists (angiotensin II, arginine vasopressin) and the adrenergic agonist phenylephrine. This suggests a link between muscle activity and growth factor production. Secretion of nerve growth factor protein by vascular smooth muscle was measured using a sensitive two-site immunoassay. Secretion is highest during muscle growth. Secretion is elevated by angiotensin II and arginine vasopressin but slightly inhibited by phenylephrine. These results suggest that cultured vascular smooth muscle can serve as a useful model in which to study the cellular regulation of trophic factor synthesis in health and disease.