Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.     

Vitronectin in the substratum of endothelial cells is cross-linked and phosphorylated.

Vitronectin (VN), previously shown to be a substrate for purified transglutaminases, was demonstrated in this study to be cross-linked when incubated with HUVEC and EAhy926 cells. The cross-linking was calcium-dependent and required that VN be plated at the substratum of the cells. These cells also phosphorylated VN, but in contrast to a previous study demonstrating a cAMP-dependent protein kinase in platelets, the phosphorylation of VN by was decreased with the addition of 1mM cAMP. The cross-linking of VN by endothelial cells demonstrates that the adhesion of these cells to VN is a dynamic process in which the substratum may be enzymatically altered. Furthermore, the modifications of VN by cross-linking and phosphorylation could modulate the functions of VN and influence events such as endothelial cell proliferation and angiogenesis.     

Stimulation of endothelial cell proliferation by vanadate is specific for microvascular endothelial cells.

Micromolar concentrations of sodium orthovanadate stimulated the proliferation of bovine capillary endothelial cells, but not bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadate treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadate was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-beta, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect at higher vanadate concentrations.     

Actin gene expression is modulated by ecdysterone in a Drosophila cell line

The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.     

Thrombospondin: synthesis and secretion by cells in culture

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.     

Bystander cell proliferation is modulated by the number of adjacent cells that were exposed to ionizing radiation.

BACKGROUND: Direct cell-to-cell contact appears to be a prerequisite for the proliferative response of bystander WB-F344 cells co-cultured with irradiated cells; however, neither gap junctional intercellular communication nor long-range factors released into the medium appear to be involved (Cytometry 2003;56A:71-80). The present work investigated whether the proliferative bystander response depends on the number of irradiated cells (cells exposed to external gamma-rays or cells exposed to short-range beta-particles emitted by DNA-incorporated (3)H-thymidine) that are adjacent to unirradiated bystander cells. METHODS: Subconfluent monolayers of rat liver epithelial cells (WB-F344) were incubated in the presence of (methyl-(3)H)thymidine at a concentration of 5.8 kBq/ml for 18 h. Radiolabeled cells containing 0.7 x 10(-3) Bq/cell (absorbed dose: 0.14 Gy) were plated together with unlabeled cells in proportions of 6% and 94%, 12% and 88%, 25% and 75%, 50% and 50%, and 75% and 25%, respectively, keeping constant the total number of plated cells. In a parallel experiment, cells acutely exposed to 5 Gy of (137)Cs gamma-rays were plated with unirradiated cells in the same proportions. In both experiments, cells were co-cultured for 24 h followed by a flow cytometric study of their proliferation. The two cell populations in the co-cultures were distinguished by staining one population with carboxyfluorescein diacetate, succinimidyl ester, which metabolizes intracellularly. RESULTS: Increasing the fraction of irradiated cells relative to unirradiated bystander cells led to an increase in proliferation of bystander cells. Specifically, in co-cultures in which irradiated cells were initially mixed with unirradiated cells in proportions of 50% and 50% and of 75% and 25%, respectively, bystander cells showed a statistically significant increase of their proliferation compared with the controls. CONCLUSIONS: The proliferative response of WB-F344 bystander cells is modulated by the number of adjacent cells that are exposed to ionizing radiation from external gamma-rays or intracellularly emitted (3)H beta-particles.     

Adhesion, shape, proliferation, and gene expression of mouse Leydig cells are influenced by extracellular matrix in vitro

Interactions between Leydig cells and the extracellular matrix (ECM) within the interstitial compartment of the mammalian testis have not been characterized. We have examined the influence of ECM on adult mouse Leydig cells by culturing the cells on different ECM substrates. Leydig cells adhere weakly to hydrated gels of type I collagen (including those supplemented with collagen types IV, V, or VIII), or to air-dried films of collagen types I, V, or VIII. In contrast, the cells attach firmly to substrates of purified type IV collagen, fibronectin, or laminin. Leydig cells also attach rapidly and adhere strongly to gelled basement membrane matrix derived from the murine Englebreth-Holm-Swarm sarcoma (Matrigel). Leydig cells assume spherical shapes and form aggregates on thick (1.5-mm) layers of Matrigel; however, on thin (0.1-mm) layers, networks of cell clusters linked by cords of elongated cells are formed within 48 h. Similar networks are formed on thick layers of Matrigel that are supplemented with type I collagen. On substrates with high ratios of collagen I to Matrigel or on untreated tissue culture plastic, Leydig cells flatten and do not aggregate. On substrates that induce rounded shapes, proliferation is inhibited and the cells maintain the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase for as long as 2 wk. Under conditions where Leydig cells are flattened, they divide and cease expressing the enzyme. Proliferating Leydig cells also exhibit elevated levels of mRNA for SPARC (Secreted Protein, Acidic and Rich in Cysteine), a Ca2(+)-binding glycoprotein associated with changes in cell shape that accompany morphogenesis and tissue remodeling. Our results indicate that the shape, association, proliferation, and expression of gene products by Leydig cells can be significantly affected in vitro by altering the composition of the extracellular substratum.     

Animal cell shape changes and gene expression.

Cell shape and cell contacts are determined by transmembrane receptor-mediated associations of the cytoskeleton with specific extracellular matrix proteins and with ligands on the surface of adjacent cells. The cytoplasmic domains of these microfilament-membrane associations at the adherens junction sites, also localize a variety of regulatory molecules involved in signal transduction and gene regulation. The stimulation of cells with soluble polypeptide factors leads to rapid changes in cell shape and microfilament component organization. In addition, this stimulation also activates the phosphoinositide signaling pathway. Recently, a linkage between actin-binding proteins and the phosphoinositide signaling pathway, was discovered. It is suggested that by the association with the second messenger system, and/or by controlling the localization of regulatory molecules, the cytoskeleton may regulate gene expression.     

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.     

Senescence of aortic endothelial cells in culture: Effects of basic fibroblast growth factor expression on cell phenotype,migration, and proliferation

Bovine aortic endothelial cells (BAEC) can be isolated in large numbers without major contamination by other cells and maintained in culture with a limited life span for about 100 population doublings. In order to study phenotypic changes of BAEC during long-term culture, stocks of different passages of BAEC were established and their morphological, migratory, and proliferative properties analyzed. Early-passage BAEC (passages 5–15) rapidly produce dense, cobblestone-like monolayers. Their growth beyond the monolayer configuration is characterized by the formation of an irregular network of spindle-shaped, crisscrossing BAEC growing either on top or beneath the monolayer, and by the assembly of elongated BAEC into well-differentiated capillary-like tubes. In contrast, senescent BAEC (passages 35–45) form perfect cobblestone monolayers that contain several, often multinucleated giant cells and a few capillary-like tubes but not the crisscrossing networks of their early-passage counterparts. The rates of BAEC migration and proliferation gradually decline during in vitro senescence. This decline is neutralized by exogenous basic fibroblast growth factor (bFGF) which elevates the migratory and proliferative capacities of early-passage and senescent BAEC to uniformly high levels. Northern blot analysis shows a gradual decline in bFGF message and an increase in laminin message during in vitro BAEC senescence. The present study supports the concept of autocrine growth regulation of BAEC and associates a decreased bFGF message with decreased rates of migration and proliferation as well as loss of the crisscrossing BAEC morphotype in senescent cultures. © 1993 Wiley-Liss, Inc.     

Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia

Vascular endothelial growth factor (VEGF) promotes neovascularization, microvascular permeability, and endothelial proliferation. We described previously VEGF mRNA and protein induction by estradiol (E2) in human endometrial fibroblasts. We report here E2 induction of VEGF expression in human venous muscle cells [smooth muscle cells (SMC) from human saphenous veins; HSVSMC] expressing both ER-alpha and ER-beta estrogen receptors. E2 at 10(-9) to 10(-8) M increases VEGF mRNA in HSVSMC in a time-dependent manner (3-fold at 24 h), as analyzed by semiquantitative RT-PCR. This level of induction is comparable with E2 endometrial induction of VEGF mRNA. Tamoxifen and hypoxia also increase HSVSMC VEGF mRNA expression over control values. Immunocytochemistry of saphenous veins and isolated SMC confirms translation of VEGF mRNA into protein. Immunoblot analysis of HSVSMC-conditioned medium detects three bands of 18, 23, and 28 kDa, corresponding to VEGF isoforms of 121, 165, and 189 amino acids. Radioreceptor assay of the conditioned medium produced by E2-stimulated HSVSMC reveals an increased VEGF secretion. Our data indicate that VEGF is E2, tamoxifen, and hypoxia inducible in cultured HSVSMC and E2 inducible in aortic SMC, suggesting E2 modulation of VEGF effects in angiogenesis, vascular permeability, and integrity.     

Differential gene expression by endothelial cells in distinct angiogenic states.

Angiogenesis is a complex process that can be regarded as a series of sequential events comprising a variety of tissue cells. The major problem when studying angiogenesis in vitro is the lack of a model system mimicking the various aspects of the process in vivo. In this study we have used two in vitro models, each representing different and distinct aspects of angiogenesis. Differentially expressed genes in the two culture forms were identified using the suppression subtractive hybridization technique to prepare subtracted cDNA libraries. This was followed by a differential hybridization screen to pick up overexpressed clones. Using comparative multiplex RT-PCR we confirmed the differential expression and showed differences up to 14-fold. We identified a broad range of genes already known to play an important role during angiogenesis like Flt1 or TIE2. Furthermore several known genes are put into the context of endothelial cell differentiation, which up to now have not been described as being relevant to angiogenesis, like NrCAM, Claudin14, BMP-6, PEA-15 and PINCH. With ADAMTS4 and hADAMTS1/METH-1 we further extended the set of matrix metalloproteases expressed and regulated by endothelial cells.     

Serotonin-induced endothelial cell proliferation is blocked by omega-3 fatty acids.

Serotonin (5HT) released from aggregating platelets at sites of vascular injury is a known mitogen for vascular endothelial cells. Recent studies have indicated that regenerating endothelial cells at sites of vessel wall injury may play a role in the development of restenosis by synthesizing and releasing growth factors for vascular smooth muscle cells, proliferation of which may result in the development of neointima. Diets rich in fish oils (omega-3 fatty acids) are associated with reduced risk of cardiovascular disease including atherosclerosis and restenosis. This study examined the effect of the omega-3 and other fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on 5HT induced endothelial cell proliferation. Among the fatty acids examined only EPA and DHA could reverse the mitogenic effect of 5HT on vascular endothelial cells, whereas oleic acid or palmitic acid did not have any effect. When added together, EPA and DHA potentiate each other in reversing the mitogenic effect of 5HT. EPA and DHA also inhibited the 5HT-induced increase in the 5HT2 receptor mRNA, without a change in the receptor density or affinity. This data suggests that one of the mechanisms by which omega-3 fatty acids may attenuate the development of atherosclerosis or restenosis is to inhibit the mitogen induced growth of vascular endothelial cells, which attenuates the release of growth factors for vascular smooth muscle cells.     

Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate

Summary Two classes of lipoxygenase (LOX) cDNAs, designated loxA and loxB, were isolated from soybean. A third lipoxygenase cDNA, loxP1, was isolated from pea. The deduced amino acid sequences of loxA and loxB show 61–74% identity with those of soybean seed LOXs. loxA and loxB mRNAs are abundant in roots and non-growing regions of seedling hypocotyls. Lower levels of these mRNAs are found in hypocotyl growing regions. Exposure of soybean seedlings to water deficit causes a rapid increase in loxA and loxB mRNAs in the elongating hypocotyl region. Similarly, loxP1 mRNA levels increase rapidly when pea plants are wilted. loxA and loxB mRNA levels also increase in wounded soybean leaves, and these mRNAs accumulate in soybean suspension cultures treated with 20 M methyl jasmonate. These results demonstrate that LOX gene expression is modulated in response to water deficit and wounding and suggest a role for lipoxygenase in plant responses to these stresses.     

Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation.

The Escherichia coli trpR gene encodes the 108-amino-acid-long Trp repressor. We have shown previously that a +1 frameshifting event occurs during the expression of trpR, resulting in the synthesis of an additional (+1 frame) polypeptide. Using trpR-lac'Z fusions, we have recently found that the transition from the 0 to the +1 frame occurs via the bypassing of a 55-nucleotide-long segment of the trpR+1-lac'Z mRNA (I. Benhar, and H. Engelberg-Kulka, Cell 72:121-130, 1993). Here we show that the frequency of trpR frameshifting (or bypassing) can be regulated both in vivo and in vitro. This frequency is inversely proportional to the rate of initiation of translation of the trpR gene. Hence, modulating the level of translation initiation affects the frequency of frameshifting.     

Thombospondin-1 disrupts estrogen-induced endothelial cell proliferation and migration and its expression is suppressed by estradiol

The natural hormone 17beta-estradiol (17beta-E2) is known to induce tumor angiogenesis in various target organs by activating positive regulators of angiogenesis. In this study, we show for the first time that in human umbilical vein endothelial cells (HUVECs), 17beta-E2 transiently down-regulates the expression and secretion of a potent negative regulator of angiogenesis, thrombospondin-1 (TSP-1). This inhibitory effect of 17beta-E2 is mediated through nongenomic estrogen receptor (ER)/mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) 1/2 and c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathways, because this effect can be abolished by a pure ER antagonist (ICI 182,780) and inhibitors of downstream signaling proteins of MAPK signaling cascades, including MAPK kinase 1/2 and ERK1/2 inhibitor and JNK/SAPK inhibitor. To understand the functional role(s) of TSP-1 during estradiol-induced angiogenesis, we examined the growth and migration of endothelial cells in different experimental environments. Using a recombinant protein, we show that increments of TSP-1 protein concentration in culture medium significantly reduce the migration and proliferation of HUVECs stimulated by 17beta-E2. Together, these studies suggest that TSP-1 can be considered an important negative factor in understanding the increased angiogenesis in response to estrogens.