Smooth muscle cell migration induced by shear-loaded platelets and endothelial cells. Enhanced platelet-derived growth factor production by shear-loaded platelets.

BACKGROUND: Uncontrolled migration and proliferation of smooth muscle cells (SMC) are the main mechanisms of development of atherosclerotic neointima. However, it has not yet been elucidated how mechanical stress is involved in this process. We investigated smooth muscle cell mitogenic activity induced by shear-loaded platelets and endothelial cells. METHODS: Experimental design: in vitro experimental study. We devised four types of conditioned media; supernatant of mixed culture of platelets and endothelial cell (ST), supernatant of shear-loaded mixed culture of platelets and endothelial cell (SH), ST medium neutralised with anti-PDGF antibody (ST+), and SH medium neutralised with anti-PDGF antibody (SH+). Smooth muscle cells were cultured in each conditioned medium, and their spreading activity was determined under a microscope. RESULTS: Smooth muscle cells spreading activity in the SH group was significantly greater than that in the ST group. Their spreading activity was suppressed by anti-PDGF antibody under shear conditions (SH+), but it was not by anti-PDGF antibody under static conditions (ST+). CONCLUSIONS: Our results demonstrate that platelet-derived growth factor is produced by shear-loaded platelets and endothelial cells, and local mechanical forces may play an important role in cardiovascular disease.     

Smooth muscle cell apoptosis in arteriosclerosis

Arteriosclerosis, a paradigmatic age-related disease, encompasses (spontaneous) atherosclerosis, restenosis after percutaneous transluminal coronary angioplasty, autologous arterial or vein graft arteriosclerosis and transplant arteriosclerosis. In all types of arteriosclerosis, vascular smooth muscle cell (SMC) accumulation in the intima is a key event, but abundant evidence also indicates the importance of SMC apoptosis in the development of arteriosclerosis. Because SMC proliferation and apoptosis coincide in arteriosclerotic lesions, the balance between these two processes could be a determinant during vessel remodeling and disease development. Various stimuli, including oxidized lipoproteins, altered hemodynamic stress and free radicals, can induce SMC apoptosis in vitro. As risk factors for arteriosclerosis, these stimuli may also lead to vascular cell apoptosis in vivo. The presence of apoptotic cells in atherosclerotic and restenotic lesions could have potential clinical implications for atherogenesis and contributes to the instability of the lesion. Based on the progress in this field, this review focuses on the mechanism and impact of SMC apoptosis in the pathogenesis of arteriosclerosis and highlights the role of biomechanical stress in SMC apoptosis.     

Heparin affinity of anionic and cationic capillary endothelial cell growth factors: analysis of hypothalamus-derived growth factors and fibroblast growth factors.

Bovine hypothalamus-derived growth factors (HDGF), pituitary fibroblast growth factor (FGF), and brain FGF were analyzed by chromatography on immobilized heparin and tested for the ability to stimulate the proliferation of capillary endothelial (CE) cells. Two distinct CE cell growth factors were found in hypothalamus, one anionic (aHDGF; pI of about 5) and one cationic (cHDGF; pI of about 8). Both aHDGF and cHDGF adhered tightly to immobilized heparin. They were eluted with 0.9-1.1 M NaCl and 1.3-1.5 M NaCl, respectively. Pituitary and brain FGF were also found to bind to immobilized heparin and to stimulate CE cell proliferation. Pituitary FGF was eluted at 1.4-1.6 M NaCl. The elution profile of brain FGF showed that two peaks of CE cell growth factor activity were eluted from the heparin column, one at 1.0 M NaCl and a second at 1.4-1.6 M NaCl. The tight binding of all of these growth factors to heparin (particularly aHDGF, whose binding is unexpected because of its negative charge) is presented as evidence that CE cell growth factors all share an affinity for heparin.     

Smooth muscle cell phenotypic switching in atherosclerosis

Smooth muscle cells (SMCs) possess remarkable phenotypic plasticity that allows rapid adaptation to fluctuating environmental cues, including during development and progression of vascular diseases such as atherosclerosis. Although much is known regarding factors and mechanisms that control SMC phenotypic plasticity in cultured cells, our knowledge of the mechanisms controlling SMC phenotypic switching in vivo is far from complete. Indeed, the lack of definitive SMC lineage-tracing studies in the context of atherosclerosis, and difficulties in identifying phenotypically modulated SMCs within lesions that have down-regulated typical SMC marker genes, and/or activated expression of markers of alternative cell types including macrophages, raise major questions regarding the contributions of SMCs at all stages of atherogenesis. The goal of this review is to rigorously evaluate the current state of our knowledge regarding possible phenotypes exhibited by SMCs within atherosclerotic lesions and the factors and mechanisms that may control these phenotypic transitions.     

Vascular endothelial growth factors and thyroid disorders

Using thyroid follicles in suspension culture, in which thyroid function is maintained for a couple of weeks, while preserving Wolff-Chaikoff effect and responding to physiological concentrations of TSH (0.1 microU/ml), we have partly elucidated the pathophysiology of increased blood flow in Graves' thyroid gland. Furthermore, we are investigating the mechanism by which iodide inhibits thyroid blood flow. Since administration of a large dose of iodide prior to subtotal thyroidectomy decreases thyroid blood flow and reduces operative morbidity and mortality in patients with Graves' disease, it is important to elucidate the mechanism by which thyroid blood flow is regulated. Furthermore, this research field will be clinically important to develop new strategies for the treatment of hypervascular and aggressive thyroid carcinoma, particularly anaplastic thyroid carcinoma. A recent report that anti-VEGF antibody reduced cancer growth in nude mice transplanted with human thyroid carcinoma is an indication this field holds for future studies.     

Vascular endothelial growth factors and liver diseases

Vascular endothelial growth factor plays an important role in neovascularization both in normal tissues and most tumors. It has been extensively investigated recently in various hepatic diseases such as primary and secondary hepatocellular carcinoma, liver cirrhosis, hepatitis and even benign tumors in liver. Vascular endothelial growth factor has been verified to be closely involved in the development and metastases of hepatocellular carcinoma and correlated to the high risk of hepatic metastases and a poor prognosis in gastrointestinal cancer. Using antibodies to vascular endothelial growth factor or other drugs to suppress its expression has also been successfully tried to restrain hepatocellular carcinoma cells and metastases in vitro and in animal models. The protein of vascular endothelial growth factor has an inclination to increase in acute and chronic hepatitis and tends to decrease in cirrhosis both in tissue expression and circulating levels. This circulating level is closely related to the Child-Pugh classification in cirrhotic liver. However, there are indeed some disagreements concerning vascular endothelial growth factor and liver disease, for example, opinions on the positive rates of vascular endothelial growth factor in protein and mRNA level are far from reaching a general consensus. Further study should be performed in the future in antitumor research and its significance in the process of liver cirrhosis.     

Platelet-derived endothelial cell growth factor inhibits DNA synthesis in vascular smooth muscle cells

Platelet-derived endothelial cell growth factor (PD-ECGF) is linked to angiogenesis in human cancer. Direct studies have demonstrated that PD-ECGF is a potent mitogen for endothelial cells in vivo. Because endothelial repair and smooth muscle cell proliferation are two processes that affect arterial wall structure and tone, we analyzed the effects of PD-ECGF on DNA synthesis and creatine kinase BB-specific activity (CK) in human umbilical artery smooth muscle cells (SMC) and in a human umbilical endothelial cell line (E304). In SMC, PD-ECGF (0.001 to 10 U/mL) inhibited DNA synthesis dose dependently (−24% + 6% to −63% + 15%) assessed by ³[H]thymidine incorporation into DNA, whereas in E304 it stimulated DNA synthesis dose dependently (30% + 4% to 100% + 4%). In both SMC and E304, however, PD-ECGF elicited an increase in CK-specific activity by 54% to 130% and 79% to 163%, respectively. These effects were reversed by a specific anti-PD-ECGF antibody. In E304 cells PD-ECGF enhanced 17β-estradiol (E₂) or dihydrotestosterone (DHT)-induced DNA synthesis from 56% to 122% and from 127% to 359%, and CK activity from 70% to 180% and from 90% to 190%, respectively. In SMC PD-ECGF, an inhibitor of ³[H]thymidine incorporation by itself, markedly enhanced the stimulatory effect of low concentrations of E₂ and DHT on ³[H]thymidine incorporation. It also increased E₂ and DHT CK induction from 40% to 140% and from 52% to 120%, respectively. In both E304 and SMC, PD-ECGF inhibited the proliferative and the CK-inducing effects of platelet-derived growth factor (PDGF) and immunoglobulin F₁ (IGF₁). Thus, PD-ECGF, an established growth promoter for endothelial cells, is a potent inhibitor of DNA synthesis in human arterial SMC. However, in both E304 endothelial cells and SMC, PD-ECGF enhances the stimulatory effect of low concentrations of gonadal steroids on ³[H]thymidine incorporation. PD-ECGF antagonizes PDGF- and IGF₁-induced DNA synthesis in both E304 and SMC cells. By inhibiting arterial SMC proliferation and accelerating endothelial cell replication, PD-ECGF may buffer the effect of PDGF and favorably modulate arterial wall response to injury.     

Smooth muscle cell hypertrophy versus hyperplasia in hypertension.

Arteries of hypertensive animals have a greater mass of smooth muscle than those of normotensive controls. We examined the contribution of smooth muscle cell hypertrophy and hyperplasia to this increase in mass. Cell size measurements obtained by (i) image analysis of enzyme-dispersed cells, (ii) morphometric evaluation of tissue sections, and (iii) biochemical measures of protein/cell and actin/cell ratios on isolated cells showed that average cell size was greater in spontaneously hypertensive rats than in normotensive Wistar-Kyoto and Sprague-Dawley controls. Average DNA/cell ratios were also increased in spontaneously hypertensive rats while protein/DNA ratios were not different. Analysis of nuclear DNA content of individual cells by flow microfluorimetry and Feulgen-DNA microdensitometry measurements showed that greater than 20% of spontaneously hypertensive rats cells were polyploid while less than 10% of control cells were polyploid. Estimates of cell number per centimeter of aortic length, based on ploidy and DNA content, show no difference between control and hypertensive rats. Thus, smooth muscle hypertrophy alone accounts for the increased mass of smooth muscle in aortas of spontaneously hypertensive rats. Furthermore, this cellular hypertrophy is accompanied by a change in nuclear ploidy. This nuclear response in hypertension may represent a fixed change related to the establishment of a chronic hypertensive state.     

Smooth muscle cell migration and proliferation: atherogenic mechanisms in hypertension

The proliferative and migratory behavior of explanted rat aortic smooth muscle cells (SMC) was investigated in cells obtained from either 24-week-old normotensive Wistar-Kyoto (WKY) or age-matched spontaneously hypertensive (SHR) rats. Time lapse video analysis of primary SMC growth in the presence of 10% serum revealed that interdivision times of cells from SHR were significantly shorter than those from WKY. Differences in the proliferative capacity of these cells were still present after two subcultivations, as analyzed by conventional growth curves. In contrast to the proliferative behavior, no differences in the migratory characteristics of SMC could be detected in a migration assay analyzing the SMC outgrowth of standardized aortic explants under low serum conditions (0.1% fetal bovine serum). It has been shown that another model of hypertension, the 4 week DOC/salt hypertensive rat results in a different reaction of SMC. Therefore, it can be considered that the extent of the potentially atherogenic alterations of SMC function in hypertension is dependent on the type, duration and the rate of increase of hypertension.     

Systems biology of vascular endothelial growth factors

Several cytokine families have roles in the development, maintenance, and remodeling of the microcirculation. Of these, the vascular endothelial growth factor (VEGF) family is one of the best studied and one of the most complex. Five VEGF ligand genes and five cell-surface receptor genes are known in the human, and each of these may be transcribed as multiple splice isoforms to generate an extensive family of proteins, many of which are subject to further proteolytic processing. Using the VEGF family as an example, we describe the current knowledge of growth-factor expression, processing, and transport in vivo. Experimental studies and computational simulations are being used to measure and predict the activity of these molecules, and we describe avenues of research that seek to fill the remaining gaps in our understanding of VEGF family behavior.     

The biology of vascular endothelial growth factors

The discovery of the vascular endothelial growth factor (VEGF) family members VEGF, VEGF-B, placental growth factor (PlGF), VEGF-C and VEGF-D and their receptors VEGFR-1, -2 and -3 has provided tools for studying the vascular system in development as well as in diseases ranging from ischemic heart disease to cancer. VEGF has been established as the prime angiogenic molecule during development, adult physiology and pathology. PlGF may primarily mediate arteriogenesis, the formation of collateral arteries from preexisting arterioles, with potential future therapeutic use in for example occlusive atherosclerotic disease. VEGF-C and VEGF-D are primarily lymphangiogenic factors, but they can also induce angiogenesis in some conditions. While many studies have addressed the role of angiogenesis and the blood vasculature in human physiology, the lymphatic vascular system has until recently attracted very little attention. In this review, we will discuss recent advances in angiogenesis research and provide an overview of the molecular players involved in lymphangiogenesis.     

Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).

The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.     

Smooth muscle cell differentiation in vitro: models and underlying molecular mechanisms

Development of in vitro models by which to study smooth muscle cell (SMC) differentiation has been hindered by some peculiarities intrinsic to these cells, namely their different embryological origins and their ability to undergo phenotypic modulation in cell culture. Although many in vitro models are available for studying SMC differentiation, careful consideration should be taken so that the model chosen fits the questions being posed. In this review, we summarize several well-established in vitro models available to study SMC differentiation from stem cells and outline novel mechanisms recently identified as underlying SMC differentiation programs.     

Smooth muscle cell transplantation into myocardial scar tissue improves heart function

This study was designed to evaluate the effect of smooth muscle cell transplantation into myocardial ventricular scar formed by cryo-necrosis. The left ventricular free wall (LVFW) of adult rats was cryo-necrosed. At 4 weeks after cryo-injury cultured fetal rat stomach smooth muscle cells (transplanted group, n = 10) or culture medium (control, n = 10) were transplanted. Sham animals (n = 8) were similarly operated but without cryo-necrosis and transplantation. The animals were administered a daily maintenance dose of cyclosporin A (5 mg/kg). At 8 weeks after cryo-injury, heart function was evaluated using a Langendorff preparation. Myocardial scar and transplanted cells were assessed histologically. Transplanted smooth muscle cells survived and formed smooth muscle cell tissue, as assessed by immunostaining against smooth muscle cell actin, within the myocardial scar. In the control hearts no muscle tissue was found in the scar. Angiogenesis occurred (P < 0.001) in the transplanted scar compared to the control scar. The transplanted cells increased the scar thickness (P < 0.01) by hyperplasia and prevented (P < 0.001) the dilatation of the ventricular chamber size compared to the controlled hearts. For physiological left ventricular volumes of 0.04 to 0.28 ml, the systolic and developed pressures in the transplanted group were greater (P < 0.001) than the control group, but less (P < 0.001) than those of the sham group. Transplanted smooth muscle cells formed smooth muscle tissue in myocardial scar tissue and improved contractile function compared to control hearts.     

Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells

Recently intimal thickening was shown to be due to stimulation of proliferation of arterial smooth muscle cells (SMC) by autocrine secretion of growth factor(s). We have reported that cultured rabbit aortic SMC secrete a growth factor (SDGF) distinct from other known growth factors. This paper reports on studies on the biological characteristics of SDGF. Putative "competence" and "progression" growth factors synergistically stimulated DNA synthesis of cultured rabbit aortic SMC. Conditioned medium (CM) from SMC containing SDGF stimulated DNA synthesis in SMC synergistically with either the competence factors or the progression factors. This synergistic effect was also observed in the presence of optimal concentrations of both competence and progression factors. The continuous presence of CM was essential for its stimulation of DNA synthesis whereas the presence of PDGF for only the first 4 h of culture was sufficient to induce maximum stimulation of DNA synthesis. These results suggest that SDGF is a new growth factor distinct from either competence or progression factors and that it stimulates a different pathway in SMC from those stimulated by other known growth factors.