The myofibroblast, cadherin, alpha smooth muscle actin and the collagen effect

In granulation tissue the myofibroblast, a specialized fibroblast characterized by cytoplasmic stress fibres with alpha smooth muscle actin (SMA), develops from mechano-tension between cells. In vitro the myofibroblast phenotype can be induced in the absence of obvious tension by plating human dermal fibroblasts at low density (LD). Upon reaching confluence the LD-plated cells express alpha SMA within stress fibres. In contrast, few cells express alpha SMA when those same fibroblasts are plated at high density (HD). Cadherins, trans-membrane proteins, and link cells tie the cytoskeletal stress fibres of neighbouring cells together. By immunohistology myofibroblasts (LD-plated fibroblasts) are shown to express cadherin-11 on their surface and between cells, while HD-plated fibroblasts expressed less cadherin-11 on their surface. Western blot analysis revealed elevated concentrations of cadherin-11 and alpha SMA in confluent LD-plated cell lysates. Reduced amounts of both proteins were found in confluent HD-plated cell lysates. Plating fibroblasts on collagen inhibits alpha SMA synthesis. When confluent LD-plated myofibroblasts were covered with a collagen lattice for 24 h, both the expression of cadherin-11 and alpha SMA were reduced by 50%. There is the possibility that direct linkage of the cytoskeleton stress fibres by cell surface cadherins maintains tension between neighbouring cells, which induces alpha SMA expression in stress fibres. Cell contact with collagen reduces cadherin expression, which may eliminate the generation of mechano-tension between fibroblasts. The other possibility is that the myofibroblast phenotype may be induced by factors other than mechano-tension.     

Regulation of Vascular Endothelial Growth Factor Expression by cAMP in Rat Aortic Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10⁻⁵M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

An anti-proliferative gene BTG1 regulates angiogenesis in vitro

B-cell translocation gene 1 (BTG1) is a member of the anti-proliferative gene family that regulates cell growth and differentiation. To clarify the role of BTG1 in angiogenesis, we examined the regulation of BTG1 expression in cultured endothelial cells and characterized its function in in vitro models of angiogenesis. BTG1 mRNA was abundantly expressed in quiescent endothelial cells. Addition of serum and angiogenic growth factors decreased BTG1 mRNA levels in endothelial cells. In contrast, BTG1 mRNA was up-regulated in tube-forming endothelial cells on Matrigel. This up-regulation was partially blocked by neutralizing antibody against transforming growth factor-beta (TGF-beta), and TGF-beta increased BTG1 mRNA levels. Inhibition of endogenous BTG1 by overexpression of antisense BTG1 resulted in inhibited network formation, and overexpression of sense BTG1 augmented tube formation in these cell lines. BTG1-overexpressing endothelial cells displayed increased cell migration. These findings suggest that BTG1 may play an important role in the process of angiogenesis.     

Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways.

Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been isolated from a variety of tumorigenic and nontransformed cell lines. Because of the importance of blood vessel growth to cell and tissue development, we have examined VEGF gene expression in a variety of mouse tissues and rodent models of cellular differentiation. Using a cloned murine VEGF cDNA we show that VEGF mRNA is expressed at relatively low levels in many adult mouse tissues examined. However, this message is dramatically induced in two models of cell differentiation: 3T3-adipose conversion and C2C12 myogenic differentiation. VEGF protein secretion is also induced in adipocyte differentiation. VEGF mRNA is markedly regulated in a pheochromocytoma (PC12) cell model of transformation and differentiation. The transformed undifferentiated cells express moderate levels of VEGF mRNA and this expression is virtually extinguished when cells differentiate into non-malignant neuron-like cells. Experiments employing phorbol esters and cAMP analogues indicate that VEGF mRNA expression is stimulated in preadipocytes by both protein kinase C and protein kinase A-mediated pathways. These results suggest that VEGF mRNA levels are closely linked to the process of cellular differentiation; they also clearly demonstrate that expression of this angiogenic factor is specifically regulated in a transformed cell line, possibly via aberrant activation of cellular second messenger pathways.     

Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo

Fisp12 was first identified as a secreted protein encoded by a growth factor-inducible immediate-early gene in mouse fibroblasts, whereas its human ortholog, CTGF (connective tissue growth factor), was identified as a mitogenic activity in conditioned media of human umbilical vein endothelial cells. Fisp12/CTGF is a member of a family of secreted proteins that includes CYR61, Nov, Elm-1, Cop-1/WISP-2, and WISP-3. Fisp12/CTGF has been shown to promote cell adhesion and mitogenesis in both fibroblasts and endothelial cells and to stimulate cell migration in fibroblasts. These findings, together with the localization of Fisp12/CTGF in angiogenic tissues, as well as in atherosclerotic plaques, suggest a possible role for Fisp12/CTGF in the regulation of vessel growth during development, wound healing, and vascular disease. In this study, we show that purified Fisp12 (mCTGF) protein promotes the adhesion of microvascular endothelial cells through the integrin receptor alphavbeta3. Furthermore, Fisp12 stimulates the migration of microvascular endothelial cells in culture, also through an integrin-alphavbeta3-dependent mechanism. In addition, the presence of Fisp12 promotes endothelial cell survival when cells are plated on laminin and deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, Fisp12 induces neovascularization in rat corneal micropocket implants. These results demonstrate that Fisp12 is a novel angiogenic inducer and suggest a direct role for Fisp12 in the adhesion, migration, and survival of endothelial cells during blood vessel growth. Taken together with the recent finding that the related protein CYR61 also induces angiogenesis, we suggest that Fisp12/mCTGF and CYR61 comprise prototypes of a new family of angiogenic regulators that function, at least in part, through integrin-alphavbeta3-dependent pathways.     

Lymphangiogenesis and biological activity ov vascular endothelial growth factor-C]

Vascular endothelial growth factor (VEGF)-C is a new member of the VEGF family, a group of polypeptide growth factors which play key roles in the physiology and pathology of many aspects of the cardiovascular system, including vasculogenesis, hematopoiesis, angiogenesis and vascular permeability. VEGF signalling in endothelial cells occurs through three tyrosine kinase receptors (VEGFRs), expressed by endothelial cells and hematopoietic precursors. With respect to the first VEGF described, VEGF-A, which is an endothelial cell specific mitogen and key angiogenic factor, VEGF-C seems to play a major role in the development of the lymphatic system. This may reflect the different binding properties of VEGFs to VEGFRs, in that VEGF-A binds to VEGFR-1 and -2, whereas VEGF-C acts through VEGFR-3, whose expression becomes restricted to lymphatics and certain veins during development. However, the finding that VEGF-C also binds to and activates VEGFR-2 may explain why it induces angiogenesis under certain conditions, which makes it relevant to experimental or clinical settings in which one would wish to block or to stimulate angiogenesis. In this paper we briefly discuss current knowledge on the biological activity of VEGF-C, emphasizing that, as has already been shown for a number of other angiogenic factors, the biological effects of VEGF-C are strictly dependent on the activity of other angiogenic regulators present in the microenvironment of the responding endothelial cells.     

CD147 depletion down-regulates matrix metalloproteinase-11, vascular endothelial growth factor-A expression and the lymphatic metastasis potential of murine hepatocarcinoma Hca-F cells

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), which is a plasma membrane glycoprotein enriched on the surface of many malignant tumors promotes adhesion, invasion and metastasis of tumor cells. In addition, tumor-associated CD147 also induces vascular endothelial growth factors (VEGFs) expression. To investigate the possible role of CD147 in the mouse hepatocarcinoma cell line Hca-F with highly metastatic potential in the lymph nodes, we used an RNA interference (RNAi) approach to silence CD147 expression. The results showed that CD147 depletion in Hca-F cells resulted in the significantly decreased expression of matrix metalloproteinase-11 (MMP-11), VEGF-A at both mRNA and protein levels. The reduced CD147 expression also attenuated the invasive, adhesive, metastatic ability of Hca-F cells to lymph nodes both in vitro and in vivo. Our current findings reveal that the tumor biological marker CD147 functionally mediates MMP-11, VEGF-A expression and tumor lymphatic metastasis.     

Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-beta

Endothelial cells express the product of the c-sis gene, which encodes the B-chain of platelet-derived growth factor (PDGF). Through local production of growth factors such as PDGF in vascular sites, endothelial cells may stimulate proliferation of adjacent cells through a paracrine mechanism. Previously, we have shown that the expression of c-sis mRNA and release of growth factor activity by human renal endothelial cells is induced by thrombin. We now show that another agent of possible importance in mediating proliferation of cells adjacent to the endothelial cell layer, transforming growth factor-beta (TGF-beta), also induced c-sis expression in these cells. In addition, we have studied the effect of agents that increase intracellular cAMP levels upon the induction of endothelial cell c-sis mRNA. The adrenergic agonists isoproterenol and norepinephrine blocked the elevation of cellular c-sis mRNA accompanying exposure to either thrombin or TGF-beta. This effect was mediated through beta-adrenergic receptors, since propranolol but not phentolamine reversed the inhibition. Forskolin, a direct activator of adenylate cyclase, also blocked induction of c-sis mRNA by thrombin and TGF-beta and inhibited the release of PDGF activity into the media of these cells. Basal, as well as stimulated c-sis mRNA levels were attenuated by these agents that increase cellular cAMP levels. These data suggest that increased cAMP production inhibits the expression of c-sis encoded mitogens by endothelial cells, and that c-sis expression is subject to bidirectional regulation in these cells.     

Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor.

Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.     

Chaotic neovascularization induced by aggressive fibrosarcoma cells overexpressing S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines essential for cell proliferation. Overexpression of S-adenosylmethionine decarboxylase in rodent fibroblasts led to aggressive transformants (Amdc-s cells) that had unforeseen high invasive capacity in nude mice, invading rapidly from the subcutaneous injection site into the peritoneal cavity and its organs. In vitro, these cells were much more invasive than Ras-oncogene-transformed fibroblasts, or human HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells. In immunohistological characterization, Amdc-s-induced tumors showed chaotic neovascularization, with abundant pleomorphic vessel-like structures that had noncontiguous or totally missing laminin (basement membrane) and CD31 (endothelial cell) immunoreactivity. Gene expression and protein analyses of Amdc-s cells showed them to overexpress several pro-angiogenic molecules, including vascular endothelial growth factor (VEGF-A), and to exhibit profound down-regulation of the anti-angiogenic thrombospondin-1 (TSP-1). By reintroduction of TSP-1 into Amdc-s cells, the high invasiveness was efficiently inhibited in vitro. Interestingly, Amdc-s cells showed up-regulation of hepatocyte growth factor (HGF) and also expressed the MET receptor, creating thus an autocrine loop able to regulate VEGF-A and TSP-1 levels. Further, we found Amdc-s cells to express increased amounts of matrix metalloproteinase-2 (MMP-2) and the large isoform of tenascin-C (TN-C), which may also contribute to the angiogenic switch and invasiveness. Consequently, Amdc-s cells offer an excellent model to sort out the key molecules of aggressive tumor growth, and thereby help in designing rational, novel anti-vascular and other cancer therapies.     

Comparison and modulation of angiogenic responses by FGFs,VEGF and SCF in murine and human fibrosarcomas

The effects of angiogenic growth factors on the growth, vascular architecture and the downstream cytokine signaling of sarcomas are unknown. These are of potential great importance since sarcoma, like endothelium, is of mesodermal origin and therefore could grow in response to these factors. Three human sarcomas (leiomyosarcoma SK-LMS-1, liposarcoma SW872 and fibrosarcoma SW684) and one murine fibrosarcoma (KHT) were grown in nude and C3H/He mice, respectively. Tumor structural vessels, perfused vessels and hypoxia were quantified immunohistochemically. Fast-growing murine KHT tumors had a markedly higher number of structural vessels compared with the human sarcomas. In both murine and human sarcomas, approximately half of the total structural vessels were perfused, and the numbers of perfused vessels decreased with increasing tumor volume. In vitro, basal mRNA expression of several angiogenic growth factors and their receptors differed between two of the human sarcoma cell lines, SK-LMS-1 and SW872. Compared with SK-LMS-1, untreated SW872 cells had higher levels of mRNA expression for FGF11, FGF14, angiopoietin, CD105 and VEGFR1. Two sarcoma cell lines were also treated with 10 ng/ml of six angiogenic growth factors (FGF1, FGF2, FGF7, FGF10, VEGF and SCF) for 24 h, and mRNA expression of endogenous FGF family members (FGF1, FGF2, FGF10, FGF11, FGF13 and FGF14) were quantitatively measured using RNase protection at various times following treatments. Again, SW872 cells were more responsive to exogenous growth factor treatment compared with SK-LMS-1 cells in terms of the elevation of endogenous FGF mRNA expression. In the SW872 cells, all of the exogenous angiogenic growth factor treatments, except for VEGF, upregulated endogenous FGF1, FGF2 and FGF14 mRNA expression. The SK-LMS-1 cells, in contrast, only responded to exogenous FGF1, FGF7 and FGF10, but did not respond to VEGF.     

The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease

Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a highly conserved receptor-binding cystine-knot structure similar to that of the platelet-derived growth factors. VEGF-A, the founding member of the family, is highly conserved between animals as evolutionarily distant as fish and mammals. In vertebrates, VEGFs act through a family of cognate receptor kinases in endothelial cells to stimulate blood-vessel formation. VEGF-A has important roles in mammalian vascular development and in diseases involving abnormal growth of blood vessels; other VEGFs are also involved in the development of lymphatic vessels and disease-related angiogenesis. Invertebrate homologs of VEGFs and VEGF receptors have been identified in fly, nematode and jellyfish, where they function in developmental cell migration and neurogenesis. The existence of VEGF-like molecules and their receptors in simple invertebrates without a vascular system indicates that this family of growth factors emerged at a very early stage in the evolution of multicellular organisms to mediate primordial developmental functions.     

The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease

Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a highly conserved receptor-binding cystine-knot structure similar to that of the platelet-derived growth factors. VEGF-A, the founding member of the family, is highly conserved between animals as evolutionarily distant as fish and mammals. In vertebrates, VEGFs act through a family of cognate receptor kinases in endothelial cells to stimulate blood-vessel formation. VEGF-A has important roles in mammalian vascular development and in diseases involving abnormal growth of blood vessels; other VEGFs are also involved in the development of lymphatic vessels and disease-related angiogenesis. Invertebrate homologs of VEGFs and VEGF receptors have been identified in fly, nematode and jellyfish, where they function in developmental cell migration and neurogenesis. The existence of VEGF-like molecules and their receptors in simple invertebrates without a vascular system indicates that this family of growth factors emerged at a very early stage in the evolution of multicellular organisms to mediate primordial developmental functions.     

Rat embryo fibroblasts transformed by c-Jun display highly metastatic and angiogenic activities in vivo and deregulate gene expression of both angiogenic and antiangiogenic factors.

The comparative tumorigenicity in rats and nude mice of cell lines derived from FR3T3 and transformed by either c-jun, ras, SV40 lt, or bovine papilloma virus type 1 (BPV1) oncogenes was investigated. c-Jun-transformed cells were as tumorigenic and metastatic as Ras-transformed cells. Latencies were short, and numerous pulmonary metastases were observed in all injected animals. In contrast, tumors induced by s.c. injection of SV40-transformed cells developed slower, and none of the animals who received injections i.v. presented with metastases. BPV1-transformed cells had an intermediate tumorigenic and metastatic activity. Microvessels present in the different tumors were revealed by immunostaining with Griffonia (Bandeiraea) Simplicifolia lectin 1. Tumors obtained with c-Jun-transformed cells exhibited more neovascularization than those induced by the other oncogenes. By comparison to FR3T3 cells or SV40- or BPV1-transformed cells, c-Jun-transformed fibroblasts repress the antiangiogenic thrombospondin-1 and SPARC genes, whereas we found that they express higher levels of gene expression of the angiogenic vascular endothelial growth factor. Finally, as compared with cells before passage in animals, thrombospondin-1, SPARC, and VEGF gene expression was also deregulated in cell lines isolated from primary tumors induced by BPV1-transformants. Our results indicate that the high transforming potential of c-Jun, evidenced as soon as transformation is established in vitro, correlates with deregulation of gene expression of both angiogenic and antiangiogenic factors leading to rapid neovascularization of tumors.