A morphometric study of vascular smooth muscle cells in culture

Summary Cultured arterial smooth muscle cells derived from different times in culture, different passages, and different species were evaluated by a combination of transmission electron microscopy and morphometry. The morphometric studies focused on point counting and monitored the following cellular components: lysosomes, myofilaments, mitochondria, ribosomes, and rough endoplasmic reticulum (RER). Percent volume composition values for the organelles involved in protein synthesis, namely ribosomes and RER, show significant fluctuations with time. Consistent with these observations, the cells showed increasing myofilaments during the early weeks in culture, which subsequently decreased significantly. The data also indicate that rabbit cells in culture may become synthetically quiescent with time and the distribution of cellular components is altered with each succeeding passage. Cultured calf (bovine) cells exhibit similar activity periods compared to rabbit but show a significantly higher lysosomal and lower myofilament content than rabbit. Calf cells could not be maintained for longer than 21 days in the absence of ascorbate, whereas ascorbate affects the ultrastructure of rabbit cells less dramatically. Age, passage, and donor, among others, are important considerations for studying in vitro smooth muscle cells. With proper morphologic and morphometric monitoring, these smooth muscle cell culture systems can be important tools in the study of aging or pathologic processes, or both. This work was presented as partial fulfillment for the degree of Ph.D. This work was supported by National Institutes of Health Grants HL-13262, HL-19717, and AG-00001.     

Collagenase‐resistant collagen promotes mouse aging and vascular cell senescence

Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1^r/r) that yields collagenase‐resistant type I collagen. Compared with wild‐type littermates, Col1a1^r/r mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1^r/r mice were susceptible to stress‐induced senescence, displaying senescence‐associated ß‐galactosidase (SA‐ßGal) activity and upregulated p16^INK4A in response to angiotensin II infusion. To elucidate the basis of this pro‐aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1^r/r or wild‐type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress‐induced SA‐ßGal activity, p16^INK4A expression, and p21^CIP1 expression. The pro‐senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3‐blocking antibody increased senescence of SMCs on wild‐type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging‐related diseases.     

Changes in proteoglycans of cultured pig aortic smooth muscle cells during subculture

Summary Smooth muscle cells were cultured from pig aorta. Changes in both the growth and the properties of sulfated proteoglycans were observed during passage. The population doubling time during log phase growth was 34 h from Passages 3 to 7–8 but 20 h at the Passage 11, and the cell density at the stationary phase, was 86 000 and 136 000 cells/cm² at Passages 3 and 11, respectively. Structural characteristics of sulfated proteoglycans secreted into the medium were investigated after metabolic labeling with [³⁵S]-sulfate. Significant differences were observed with age in vitro: a) [³⁵S]proteoglycan complexes were in a greater amount at Passage 10 than at Passage 3; b) the hydrodynamic size of at least 45% of subunits and about 90% of monomers decreased with in vitro aging; c) this decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains; d) an increase of 15% in the proportion of dermatan sulfate was observed when cells were subjected to 10 passages. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM, U. 181) and the Fondation pour la Recherche Médicale.     

Modulation of DNA synthesis in aortic smooth muscle cells by dinitrotoluenes

Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced ³H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1–100 µM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1–100 µM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions.Abbreviation DAT diaminotuluene - tDNT technical grade dinitrotoluene - DNT dinitrotoluenes - HU hydroxyurea - IP intraperitoneal - LDH lactate dehydrogenase - MCT oil medium chain triglyceride - NPTC non-protein thiol content - RDS replicative DNA synthesis - SEM standard error of the mean - SMC smooth muscle cells - UDS unscheduled DNA synthesis     

The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.     

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.     

Transforming growth factor-beta: Signal transduction via protein kinase C in cultured embryonic vascular smooth muscle cells

Summary Transforming growth factor-beta (TGF-β), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-α exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-β on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-β increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-β had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-β potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-β are heterogeneous and are functions of the embryonic lineage of the VSMC.     

Visualization of stimulus‐specific heterogeneous activation of individual vascular smooth muscle cells in aortic tissues

Intercellular communication among autonomic nerves, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs) plays a central role in an uninterrupted regulation of blood flow through vascular contractile machinery. Impairment of this communication is linked to development of vascular diseases such as hypertension, cerebral/coronary vasospasms, aortic aneurism, and erectile dysfunction. Although the basic concept of the communication as a whole has been studied, the spatiotemporal correlation of ECs/VSMCs in tissues at the cellular level is unknown. Here, we show a unique VSMC response to ECs during contraction and relaxation of isolated aorta tissues through visualization of spatiotemporal activation patterns of smooth muscle myosin II. ECs in the intimal layer dictate the stimulus‐specific heterogeneous activation pattern of myosin II in VSMCs within distinct medial layers. Myosin light chain (MLC) phosphorylation (active form of myosin II) gradually increases towards outer layers (approximately threefold higher MLC phosphorylation at the outermost layer than that of the innermost layer), presumably by release of an intercellular messenger, nitric oxide (NO). Our study also demonstrates that the MLC phosphorylation at the outermost layer in spontaneously hypertensive rats (SHR) during NO‐induced relaxation is quite high and approximately 10‐fold higher than that of its counterpart, the Wister–Kyoto rats (WKY), suggesting that the distinct pattern of myosin II activation within tissues is important for vascular protection against elevated blood pressure.     

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.     

Chemical induction of cellular antioxidants affords marked protection against oxidative injury in vascular smooth muscle cells

Extensive evidence suggests that reactive oxygen species are critically involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis and myocardial ischemia-reperfusion injury. Consistent with this concept, administration of exogenous antioxidants has been shown to be protective against oxidative cardiovascular injury. However, whether induction of endogenous antioxidants by chemical inducers in vasculature also affords protection against oxidative vascular cell injury has not been extensively investigated. In this study, using rat aortic smooth muscle A10 cells as an in vitro system, we have studied the induction of cellular antioxidants by the unique chemoprotector, 3H-1,2-dithiole-3-thione [corrected] (D3T) and the protective effects of the D3T-induced cellular antioxidants against oxidative cell injury. Incubation of A10 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants in a concentration-dependent manner. These included reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase, superoxide dismutase, and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, A10 cells were pretreated with D3T and then exposed to either xanthine oxidase (XO)/xanthine, 4-hydroxynonenal, or cadmium. We observed that D3T pretreatment of A10 cells led to significant protection against the cytotoxicity induced by XO/xanthine, 4-hydroxynonenal or cadmium, as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium reduction assay. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in vascular smooth muscle cells can be induced by exposure to D3T, and that this chemical induction of cellular antioxidants is accompanied by markedly increased resistance to oxidative vascular cell injury.     

培养大鼠主动脉血管平滑肌细胞产生巨噬细胞集落刺激因子及其受体的表达

本研究用培养大鼠主动脉血管平滑肌细胞（ＶＳＭＣｓ）,结果如下：（１）用生物活性检测法发现ＶＳＭＣｓ无血清条件培养液可刺激巨噬细胞集落形成,其作用能被抗巨噬细胞集落刺激因子（ＭＣＳＦ）抗体抑制;（２）用免疫细胞化学技术证明ＶＳＭＣｓ存在ＭＣＳＦ受体;（３）用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ技术证明ＶＳＭＣｓ有ＭＣＳＦ及其受本的ｍＲＮＡ表达,血清刺激使两者表达明显增强。本研究首次报道了培养大鼠主动脉ＶＳＭＣ     

Growth of aortic vascular smooth muscle cells in lowered oxygen tension

Summary Primary cultures of vascular smooth muscle cells, isolated from rat aorta, were grown under normoxic (20% O₂) and mildly hypoxic (5 % O₂) conditions. Cells from both conditions were compared for growth characteristics, morphology, protein synthesis, lysosomal enzyme activity, and oxygen consumption. In no case was a consistently significant difference observed. These observations indicate that these cells can adapt or are adapted to mildly hypoxic conditions. Moreover, these results may indicate that the culture of vascular smooth muscle cells in mild hypoxia represents a closer approximation of in vivo growth conditions for these cells.Supported by HL19242     

一氧化氮在吲哚昔酚抑制血管平滑肌细胞增殖中的作用

目的：观察吲哚昔酚（ldoxifene,ldo）对大鼠血管平滑肌细胞增殖的影响,并探讨平滑肌源性一氧化氮（NO）在其中的作用。方法：血管平滑肌细胞培养、NO释放的测定、细胞计数和MTT测定。结果：吲哚昔酚可剂量依赖性的促使血管平滑肌细胞NO的释放,10μmol／L吲哚昔酚明显抑制10％胎牛血清（FCS）和10^-7mol／L的ET-1诱导的细胞增殖,吲哚昔酚的抑制作用可被一氧化氮合酶抑制剂L-NAME（100μmol／L）和鸟苷酸环化酶（guanylate cyclase,GC）抑制剂美蓝（methylene blue,MB）（10μmol／L）明显减轻。结论：吲哚昔酚抑制血管平滑肌细胞增殖的作用与其NO释放密切相关,其中可能有NO-GC-cGMP通路的参与。     

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF-BB-induced rat aortic VSMCs proliferation and [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin-dependent kinase inhibitor (CKI) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta-receptor, extracellular signal-regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF-BB-induced rat aortic VSMCs proliferation via G(0)/G(1) arrest in association with modulation of the expression or activation of cell-cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system.     

Restoration of CPEB4 prevents muscle stem cell senescence during aging

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