Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line.

A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of approximately 1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction. This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with blood-vessel growth and maintenance.     

Mesenchymal target cell specificity of egg granuloma-derived fibroblast growth factor in schistosomiasis

We previously demonstrated that egg granulomas isolated from the liver of mice infected with Schistosoma mansoni secrete factors that stimulate fibroblast proliferation in vitro. Because this growth factor also stimulated thymocyte proliferation, we further investigated the mesenchymal target cell specificity of this factor. We now report that granulomas also secrete material that promotes growth of aortic smooth muscle and endothelial cells. We found that the activities that stimulate fibroblast and smooth muscle cell growth share similar physicochemical properties (Mr, 30-40 kilodaltons [kDa]; pI, approximately 6.5), a result suggesting that they are the same or related molecules. In contrast, fractions (Mr, less than 10 kDa; pI, 6.7-7.5) that maximally stimulated endothelial cells had minimal or no effects on the other two cell types. We postulate that soluble granuloma products might also stimulate in vivo proliferation of vascular smooth muscle and endothelial cells and contribute to the pathology of the intrahepatic vessels in schistosomiasis.     

Purinergic signaling and vascular cell proliferation and death

Evidence for the role of purinergic signaling (via P1 and P2Y receptors) in the proliferation of vascular smooth muscle and endothelial cells is reviewed. The involvement of the mitogen-activated protein kinase second-messenger cascade in this action is clearly implicated, although details of the precise intracellular pathways involved still remain to be determined. Synergistic actions of purines and pyrimidines with growth factors occur in promoting cell proliferation. Interaction between purinergic signaling for vascular cell proliferation and cell death mediated by P2X7 receptors is discussed. There is evidence of the release of ATP from endothelial cells, platelets, and sympathetic nerves as well as from damaged cells in atherosclerosis, hypertension, restenosis, and ischemia; furthermore, there is evidence that vascular smooth muscle and endothelial cells proliferate in these pathological conditions. Thus, the involvement of ATP and its breakdown product, adenosine, is implicated; it is hoped that with the development of selective P1 (A2) and P2Y receptor agonists and antagonists, new therapeutic strategies will be explored.     

Angiotensin II and cell cycle regulation

Angiotensin II has emerged as an important growth factor for vascular, cardiac, and renal cells. Depending on the specific cell type and presence of other growth factors, angiotensin II induces proliferation (replication of DNA with subsequent successful division of cells), hypertrophy (increase in cell size, cell protein, and mRNA content without DNA replication), apoptosis (programmed cell death), or differentiation. Such angiotensin II-mediated modulation of growth process may underlie various pathophysiological processes such as atherosclerosis, vascular and cardiac remodeling, and progression of chronic renal disease. Clearly, angiotensin II-induced proliferation requires complete cell progression through the various steps of the cell cycle. In contrast, cells undergoing angiotensin II-mediated hypertrophy are arrested in the G1-phase. Upregulation of cell cycle-dependent kinase inhibitors (eg, p27Kip1) plays an important role in this process. Although accumulating evidence suggests that apoptosis is cell cycle-dependent, only few data are currently available concerning the interaction of angiotensin II with the cell cycle machinery in apoptosis. We review the various angiotensin II-mediated growth processes and their relationship to events governing cell cycle regulation.     

PDGF通过AKT/FoxO信号通路促进血管平滑肌细胞增殖

目的探讨血小板源生长因子(PDGF)促进血管平滑肌细胞增殖的分子机制方法用PDGF处理体外培养的大鼠动脉平滑肌细胞A10,MTT检测TPDGF对平滑肌细胞增殖的影响;用Western Blot检测AKT、FoxO1/3a的总蛋白和磷酸化水平。结果 PDGF可促进平滑肌细胞的增殖,且呈明显的剂量恶化时间依赖关系。PDGF可明显升高AKT和FoxO1/3a的磷酸化水平,呈时间依赖的关系。PI3K抑制剂可阻断PDGF促进平滑肌细胞的增殖和增高AKT和FoxO1/3a磷酸化的作用,但MEK和JNK信号通路抑制剂无明显作用。结论 PDGF通过激活AKT/FoxO1/3a信号途径促进血管平滑肌细胞的增殖。     

Bone marrow-derived stem cell interactions with adult cardiomyocytes and skeletal myoblasts in vitro

BACKGROUND AND OBJECTIVES: Secreted growth factors and cell-to-cell contact are both required to elicit cellular functions. We tested the hypothesis that bone-marrow-derived growth factors, together with cell-to-cell contact between bone-marrow-derived stem cells and cardiomyocytes or myoblasts, promote the proliferation of cardiomyocytes and myoblasts. METHODS: Human cardiomyocytes or skeletal myoblasts were cultured for 4 days in the presence of low and high concentrations of bone-marrow-derived mononuclear cell conditioned medium (MNC-CM) or marrow stromal cell conditioned medium (MSC-CM). The concentrations of vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 in their respective conditioned media were assayed by enzyme-linked immunosorbent assay. Stem cells were mixed with cardiomyocytes or skeletal myoblasts at a 1:1 ratio and cultured for 7 days to assess the proliferation of these cells. In parallel experiments, equal numbers of various cell types were cultured alone. RESULTS: The concentrations of VEGF, MCP-1, and HGF increased in MNC-CM and MSC-CM. MNC-CM showed no effect on cardiomyocyte proliferation. A low concentration of MSC-CM increased cardiomyocyte proliferation by 60% (P<.05). Low concentrations of MNC-CM or MSC-CM showed a trend toward an increased proliferation of myoblasts. A high concentration of either conditioned medium showed a toxic effect. In contact coculture, the proliferation of cardiomyocytes and MNC showed no synergistic effect; instead, there was some evidence of inhibition. The proliferation of cardiomyocytes and stromal cells showed an additive effect. Myoblasts in contact coculture with MNC or MSC showed no synergistic effect. CONCLUSION: These in vitro results suggest that paracrine effects may be the mechanism by which stromal cells become beneficial in cardiac therapy. MNC do not induce the proliferation of cardiomyocytes. Stem-cell-secreted growth factors induce the proliferation of myoblasts, which is not influenced by cell-to-cell contact.     

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.     

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.     

Do plasma and serum have different abilities to promote cell growth?

The abilities of plasma and serum to support the growth of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of fibroblast growth factor (FGF) incrases the growth rate of the cultures in both cases. In contrast, when vascular smooth muscle cells are maintained on an ECM, they proliferate equally well exposed to either plasma or serum. Because the cultures had an average doubling time (15 hr) that was already at a minimum, FGF no longer had an effect on vascular smooth muscle cell proliferation. These results raise the possibility that the lack of response of vascular smooth muscle cells, as well as that of other cell types in vitro, to plasma factors is not an intrinsic property of the cells but is rather due to the substrate upon which the cells rest. Because cells maintained on an ECM respond to plasma factors, it is likely that the close contact of the cells with the ECM restores their sensitivity to physiological factors present in plasma.     

A role for planar cell polarity signaling in angiogenesis

The planar cell polarity (PCP) pathway is a highly conserved signaling cascade that coordinates both epithelial and axonal morphogenic movements during development. Angiogenesis also involves the growth and migration of polarized cells, although the mechanisms underlying their intercellular communication are poorly understood. Here, using cell culture assays, we demonstrate that inhibition of PCP signaling disrupts endothelial cell growth, polarity, and migration, all of which can be rescued through downstream activation of this pathway by expression of either Daam-1, Diversin or Inversin. Silencing of either Dvl2 or Prickle suppressed endothelial cell proliferation. Moreover, loss of p53 rescues endothelial cell growth arrest but not the migration inhibition caused by PCP disruption. In addition, we show that the zebrafish Wnt5 mutant (pipetail (ppt)), which has impaired PCP signaling, displays vascular developmental defects. These findings reveal a potential role for PCP signaling in the coordinated assembly of endothelial cells into vascular structures and have important implications for vascular remodeling in development and disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anti-endothelial cell IgG from patients with chronic arsenic poisoning induces endothelial proliferation and VEGF-dependent angiogenesis

An endemic peripheral vascular disorder due to chronic arsenic poisoning, named Blackfoot disease (BFD), occurs in Taiwan. BFD causes destruction of vascular endothelial cells, and an anti-endothelial cell IgG antibody was found in the sera of BFD patients. We studied the role of this IgG antibody (BFD-IgG) in modulating proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and found that a low concentration of BFD-IgG (200 μg/mL) stimulated endothelial cell growth and increased expressions of vascular cell adhesion molecule-1 (VCAM-1), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF). The apoptosis events appeared not altered by addition of BFD-IgG. An in vitro neoangiogenesis assay demonstrated that BFD-IgG promoted the formation of tube-like structures, which was completely abrogated by anti-VEGF neutralizing antibody and partially by NOS inhibitor, L-NAME. We conclude that BFD-IgG at 200 μg/mL results in cell proliferation and enhanced VEGF-dependent angiogenesis in vitro. Those results suggested that a low concentration of BFD-IgG plays a protective role in the pathogenesis or the progression of BFD.     

Role of transforming growth factor-alpha in von Hippel--Lindau (VHL)(-/-) clear cell renal carcinoma cell proliferation: a possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL(-/-) RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL(-/-) RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-alpha (TGF-alpha), another VHL-regulated growth factor. We show that TGF-alpha mRNA and protein are hypoxia-inducible in VHL(-/-) RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL(-/-) RCC cells, TGF-alpha can also act as a specific growth-stimulatory factor for VHL(-/-) RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL(-/-) RCC cells, such as vascular endothelial growth factor and TGF-beta1, which do not stimulate RCC cell proliferation. A TGF-alpha-specific antisense oligodeoxynucleotide blocked TGF-alpha production in VHL(-/-) RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL(-/-) RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-alpha stimulates proliferation. These results suggest that the generation of a TGF-alpha autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.     

TGF-β effects on airway smooth muscle cell proliferation, VEGF release and signal transduction pathways

Background and objective:   Airway smooth muscle (ASM) cell hyperplasia is a key feature of airway remodelling. Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) are key components in signal transduction associated with cell proliferation; MAPK consists of the extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun NH₂-terminal kinase (JNK). The effect of transforming growth factor (TGF)-β on the proliferation of ASM cells, the release of vascular endothelial growth factor (VEGF) by ASM cells and relevant signal transduction pathways were investigated.
Methods:   ASM cells were growth-arrested for 48 h then stimulated with platelet-derived growth factor (PDGF), TGF-β and dexamethasone. ASM cells were also treated with specific inhibitors of MAPK (PD98059), PI3K (wortmannin) and JNK (SP600125). Cell proliferation and VEGF concentrations were measured.
Results:   TGF-β neither augmented ASM cell proliferation nor showed a synergistic effect on PDGF-mediated ASM cell proliferation. Dexamethasone did not suppress ASM cell proliferation. VEGF release was augmented by TGF-β stimulation in a time-dependent manner, and was further enhanced by co-stimulation with PDGF and TGF-β. Dexamethasone suppressed VEGF release significantly. TGF-β enhanced PI3K phosphorylation, while PDGF augmented both ERK and PI3K phosphorylation. Wortmannin inhibited both TGF-β- and PDGF-stimulated VEGF release.
Conclusions:   TGF-β may facilitate airway remodelling by promoting VEGF release through the PI3K pathway, rather than via ASM cell proliferation.     

Coordination of plant cell division and expansion in a simple morphogenetic system

Morphogenesis in plants arises from the interplay of genetic and physical interactions within a growing network of cells. The physical aspects of cell proliferation and differentiation are genetically regulated, but constrained by mechanical interactions between the cells. Higher plant tissues consist of an elaborate three-dimensional matrix of active cytoplasm and extracellular matrix, where it is difficult to obtain direct measurements of geometry or cell interactions. To properly understand the workings of plant morphogenesis, it is necessary to have biological systems that allow simple and direct observation of these processes. We have adopted a highly simplified plant system to investigate how cell proliferation and expansion is coordinated during morphogenesis. Coleocheate scutata is a microscopic fresh-water green alga with simple anatomical features that allow for accurate quantification of morphogenetic processes. Image analysis techniques were used to extract precise models for cell geometry and physical parameters for growth. This allowed construction of a deformable finite element model for growth of the whole organism, which incorporated cell biophysical properties, viscous expansion of cell walls, and rules for regulation of cell behavior. The study showed that a simple set of autonomous, cell-based rules are sufficient to account for the morphological and dynamic properties of Coleochaete growth. A variety of morphogenetic behavior emerged from the application of these local rules. Cell shape sensing is sufficient to explain the patterns of cell division during growth. This simplifying principle is likely to have application in modeling and design for engineering of higher plant tissues.     

Novel cardiovascular actions of the activins.

Proliferation and directed migration of vascular cells are key components in vascular diseases such as atherosclerosis and restenosis following percutaneous transluminal coronary angioplasty. However, the precise cellular and molecular mechanisms involved in the control of vascular cell proliferation or migration at the tissue level remain largely undefined. Molecules contributing to these processes are elaborated by distinct cell types and act in both autocrine and paracrine modes. They include two broad classes, polypeptide growth factors and vasoactive G-protein-coupled receptor (GPCR) agonists. Examples of the former, such as platelet-derived growth factor, bind to and activate cell surface receptor tyrosine kinases, initiating intracellular biochemical signaling pathways associated with cell proliferation or migration. In contrast, recent evidence suggests that vasoactive GPCR agonists (e.g. angiotensin II, endothelin-1, alpha-thrombin) elicit cell growth indirectly by inducing the production of autocrine or paracrine factors in vascular cells. Recent studies have identified activin A as a novel component of conditioned medium obtained from GPCR agonist-stimulated vascular smooth muscle cells (SMCs). Although activin A alone only weakly stimulated rat aortic SMC DNA synthesis, it demonstrated a potent co-mitogenic effect in combination with either epidermal growth factor (EGF) or heparin binding EGF-like growth factor in these cells, increasing DNA synthesis by up to 5- and 4-fold respectively. Furthermore, in a rat carotid-injury model, activin A mRNA was upregulated within 6 h after injury, followed by increases in immunoreactive protein detected in the expanding neointima 7 to 14 days later. Taken together, these results indicate that activin A is a common vascular SMC-derived growth factor induced by vasoactive agonists that may, either alone or in combination with other factors, contribute to fibroproliferative vascular diseases.     

Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation

Atherosclerosis, in its myriad incarnations the foremost killer disease in the industrialized world, is characterized by aberrant proliferation of vascular smooth muscle (VSM) cells in part as a result of the recruitment of inflammatory cells to the blood vessel wall. The epoxyeicosatrienoic acids are synthesized from arachidonic acid in a reaction catalyzed by the cytochrome P450 system and are vasoactive substances. Metabolism of these compounds by epoxide hydrolases results in the formation of compounds that affect the vasculature in a pleiotropic manner. As an outgrowth of our observations that urea inhibitors of the soluble epoxide hydrolase (sEH) reduce blood pressure in spontaneously hypertensive rats as well as the findings of other investigators that these compounds possess antiinflammatory actions, we have examined the effect of sEH inhibitors on VSM cell proliferation. We now show that the sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) inhibits human VSM cell proliferation in a dose-dependent manner and is associated with a decrease in the level of cyclin D1. In addition, cis-epoxyeicosatrienoic acid mimics the growth-suppressive activity of CDU; there is no evidence of cellular toxicity or apoptosis in CDU-treated cells when incubated with 20 microM CDU for up to 48 h. These results, in light of the antiinflammatory and antihypertensive properties of these compounds that have been demonstrated already, suggest that the urea class of sEH inhibitors may be useful for therapy for diseases such as hypertension and atherosclerosis characterized by exuberant VSM cell proliferation and vascular inflammation.     

Update on tumor cell procoagulant factors

Tumor cells produce tissue factor, cancer procoagulant, plasminogen activators and other factors that interact with the coagulation system, the fibrinolytic system and vascular or blood cells such that they can upset the normal homeostasis and balance between activation and inhibition of the coagulation and fibrinolytic systems. These activities play a role in tumor cell growth and metastasis, vascular wall function, and hemostasis. Proteases and their inhibitors are intimately involved in all aspects of the hemostatic, cell proliferation and cellular signalling systems. This review provides a brief examination of recent observations in this complex interaction of cellular and hemostatic factors.     

Paracrine effect of human vascular endothelial cells on human vascular smooth muscle cell proliferation: Transmembrane co-culture method

Summary To investigate the role endothelial cells have on underlying smooth muscle cell proliferation, human aortic vascular smooth muscle cells were co-cultured with human aortic endothelial cells at different cell densities, using a transmembrane co-culture method. Subconfluent endothelial cells subseeded at low (0.5 × 10⁴ cells/well) and medium densities (2.0 × 10⁴ cells/well) stimulated smooth muscle cell proliferation by 43 ± 14% (P < 0.01) and 39 ± 8% (P < 0.02), respectively. However, this stimulatory effect on smooth muscle cell proliferation was not evident in confluent endothelial cells subseeded at high cell density (8.0 × 10⁴ cells/well). Treatment of smooth muscle cells with trapidil, at 10^–6M, for anti-platelet derived growth factor (PDGF) effect or with endothelin-1 receptor blocker FR 139317, at 10^–6M failed to inhibit this stimulatory effect. These results imply that subconfluent human endothelial cells are able to exert a stimulatory effect on human smooth muscle cell proliferation, and that this endothelial paracrine growth effect may not be mediated by endothelin or PDGF.     

Mechanisms of telomerase induction during vascular smooth muscle cell proliferation

Telomeres are primarily controlled by a highly specialized DNA polymerase termed telomerase. Recent studies have demonstrated that introduction of the telomerase catalytic component (TERT) into telomerase-negative cells activates telomerase and extends cell life span, whereas mice lacking telomerase activity revealed impaired cell proliferation in some organs as well as reduced tumorigenesis. These reports suggest that telomerase plays an important role in long-term cell viability and cell proliferation. However, the mechanism or mechanisms by which telomerase is induced or regulated remains to be elucidated. We report here that primary vascular smooth muscle cells (VSMCs) express telomerase and that increased telomerase activity correlates with cell proliferation. Inhibition of telomerase diminished growth of VSMCs, which suggests a crucial role for telomerase activation in the regulation of VSMC proliferation. We propose a novel model whereby telomerase is first activated in the cytoplasm before cell proliferation, followed by accumulation of activity in the nucleus during the logarithmic phase of cell growth. Activation of telomerase in VSMCs was linked to phosphorylation of TERT. The protein kinase inhibitor H7 suppressed the activation of telomerase in the cytoplasm and also inhibited the accumulation of TERT as well as telomerase activity in the nucleus. These data suggest that posttranslational modification of TERT by phosphorylation is important for activation and accumulation of telomerase into the nucleus in the process of VSMC proliferation.