Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture.

Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 ng/ml of HGF at rates comparable to that of 20 ng/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor-1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of EGF.     

A novel biological effect of atrial natriuretic hormone: inhibition of mesangial cell mitogenesis

We have investigated the effect of atrial natriuretic hormone on serum-induced mitogenesis in cultured rat mesangial cells. Synthetic peptides, atriopeptin 28 and atriopeptin 24, dose-dependently decreased thymidine incorporation, with a half-maximal effect at approximately 1 nM and a maximal inhibition of approximately 60%. Moreover, atriopeptin 28 significantly decreased the clonal proliferation of mesangial cells. Atriopeptin 28 also decreased resting cytosolic Ca but had no effect on the increase induced by serum, relative to the lower baseline established by atriopeptin 28. Nevertheless, the overall effect of atriopeptin 28 on Ca was to attenuate the serum-induced increase, relative to the original resting level. These results therefore provide evidence for a novel biological effect of atrial natriuretic hormone and suggest that the antimitogenic effect may be mediated by atriopeptin-induced alterations of intracellular Ca dynamics. We speculate that atrial natriuretic hormone may be a modulator of mesangial cell mitogenesis in vivo.     

Nitric oxide-generating vasodilators inhibit mitogenesis and proliferation of BALB/C 3T3 fibroblasts by a cyclic GMP-independent mechanism.

The purpose of this study was to investigate the effects of nitric oxide-generating vasodilators and 8-bromo-cGMP on serum-induced mitogenesis in BALB/c 3T3 fibroblasts that lack soluble guanylate cyclase activity. Two such vasodilators, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate, decreased the incorporation of (3H)thymidine in these cells dose-dependently whereas 8-bromo-cGMP was ineffective at concentrations of up to 10 mM. Moreover, S-nitroso-N-acetylpenicillamine also inhibited cell proliferation, consistent with the data on (3H)thymidine incorporation. S-nitroso-N-acetylpenicillamine had no effect on cGMP accumulation, confirming previous studies that these cells lack soluble guanylate cyclase activity. Hemoglobin and FeSO4/ascorbate, agents that inhibit the actions of nitric oxide, both decreased S-nitroso-N-acetylpenicillamine-induced antimitogenesis, supporting the view that this effect was related to the generation of nitric oxide. The antimitogenic activity of S-nitroso-N-acetylpenicillamine was unlikely to be the expression of nitric oxide-induced degradation of serum mitogens, as indicated by the decrease of the antimitogenic activity on prolonged preincubation of SNAP in serum-containing medium. We conclude that nitric oxide-generating vasodilators inhibit serum-induced mitogenesis and cell proliferation in BALB/c 3T3 fibroblasts by a cGMP-independent mechanism.     

On the mechanisms of inhibition of lymphocyte mitogenesis by high conA concentrations

ConA at high concentrations inhibits lymphocyte mitogenesis. Previous studies have shown that inhibitory conA concentrations do not inhibit the acquisition of responsiveness to interleukin-2 (IL-2) when excessive conA is removed. To analyse further the problem of high-dose inhibition by conA, we determined whether inhibition of mitogenesis is related to inhibition of IL-2 production or, alternatively, whether factor production is intact, but the cells are rendered incapable of responding to the factor. ConA stimulates IL2 production at concentrations that are inhibitory to mitogenesis of human lymphocytes. IL-2 was assayed both in a murine cytotoxic T cell line and human memory cells. The response of IL-2-dependent cells to IL-2-containing medium was, on the other hand, inhibited by conA in a dose-dependent fashion. One mechanism whereby high conA concentrations inhibit mitogenesis is by rendering cells resistant to IL-2, possibly via extensive cross-linking of cell surface sites.     

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.     

Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP

We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 microM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine) which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP.     

Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells.

Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells.     

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.     

Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells.

The current studies were designed to explore the effects of C-receptor-binding atrial natriuretic peptide analogues on serum-induced mitogenesis in cultured rat aortic smooth muscle cells. To this end, rANF99-126 and a series of truncated (rANF103-126, rANF103-125), ring-deleted (des[Gln116, Ser117, Gly118, Leu119, Gly120]rANF102-121-NH2 (c-ANF) and linear des(Cys105, Cys121)rANF104-126 peptide analogues were used. The latter two peptides have been reported to be selective for the ANF-C receptor. In cells subcultured between passage 3 to 19, rANF99-126, rANF103-126, and rANF103-125 concentration-dependently (0.1-1000 nM) inhibited serum-induced (3H) thymidine incorporation with maximal inhibition observed at 1 microM for each peptide (approximately 40, 31 and 56%) respectively. Furthermore, des[Cys105, Cys121]rANF104-126 inhibited serum-induced (3H)thymidine incorporation concentration-dependently without altering basal or elevated cellular cAMP or cGMP levels. Moreover, the reduction in thymidine incorporation was associated with inhibition of serum-induced clonal cell proliferation. In contrast, c-ANF failed to inhibit serum-induced mitogenesis, yet at a concentration of 100 nM it antagonized the antimitogenic effects of des[Cys105, Cys121]rANF104-126 or rANF99-126 without having any effect on basal or elevated cellular cyclic nucleotide levels. We conclude that the antimitogenic effect of atrial peptides is mediated through interaction with the ANF-C receptor and may be independent of changes in cellular cyclic nucleotide levels.     

Decreased response to epidermal growth factor during cellular senescence in cultured human microvascular endothelial cells.

We have previously demonstrated that epidermal growth factor (EGF) induces cell migration, tissue-type plasminogen activator synthesis, as well as tubular formation in microvascular endothelial cells from human omental tissue. In this study, we compared the responsiveness to EGF of late passaged (senescent) human omental microvascular endothelial (HOME) cells with that of early passaged (young) HOME cells. We have employed HOME cells derived from surgically resected omental samples from 14 patients. EGF-stimulated cell migration significantly more in the young cells than in the senescent cells during serial cultivation (aging) in vitro. Scatchard analysis demonstrated that the number for both high and low affinity receptors for EGF in HOME cells was decreased dramatically during serial cultivation. The expression of EGF receptor mRNA was also decreased in the senescent HOME cells. Treatment of HOME cells with EGF significantly increased cellular mRNA levels of tissue-type plasminogen activator, and two protooncogenes, c-fos and c-myc, in young HOME cells, but not in senescent HOME cells. Thus HOME cells aged in vitro show a decreased responsiveness to EGF, resulting in decreased migration of human endothelial cells. The serial cultivation of human endothelial cells in vitro may downregulate EGF receptor and decrease responsiveness to exogenous EGF, a potent angiogenic factor.     

Phospholipase C-γ1 is required for the epidermal growth factor receptor-induced squamous cell carcinoma cell mitogenesis

The epidermal growth factor receptor (EGFR) is a key driver in the process of squamous cell carcinoma (SCC) cell mitogenesis. Phospholipase C-γ1 (PLC-γ1) is a downstream target of EGFR signaling, but the role and necessity of PLC-γ1 in EGFR-induced cell mitogenesis remain unclear. In the present study, we report an elevated expression of PLC-γ1 in human SCC biopsies relative to adjacent normal epidermis, and in human SCC cell lines compared to normal human keratinocytes. EGFR-induced SCC cell mitogenesis was blocked by small interfering RNA knockdown of PLC-γ1. However, inhibition of the catalytic activity of phospholipase C had no effect on EGFR-induced SCC cell mitogenesis. In response to the EGFR ligand epidermal growth factor (EGF), PLC-γ1 was translocated not only to the plasma membrane but also to the nucleus. These data suggest that PLC-γ1 is required for EGFR-induced SCC cell mitogenesis and the mitogenic function of PLC-γ1 is independent of its lipase activity.     

Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

Identification of an endothelial cell growth-inhibitory activity produced by human monocytes

The control of endothelial cell proliferation is important in a variety of processes including wound healing and tumor-induced angiogenesis. We have observed that normal unstimulated human monocytes isolated from the blood can inhibit human endothelial cell proliferation. Monocyte-conditioned medium was fractionated by gel filtration chromatography, yielding a 175-fold enrichment of a growth inhibitory activity, designated monocyte-derived endothelial cell inhibitory factor (MECIF). MECIF was found to be protease sensitive, resistant to acid treatment, and heat labile. When conditioned medium was subjected to HPLC gel filtration, the inhibitory activity was eluted as a single peak with a molecular weight of 50-70 kDa. Several characteristics distinguish MECIF from previously described monocyte/macrophage-derived inhibitory factors. Unlike TGF-beta, MECIF is heat labile and does not induce a mitogenic response in growth-arrested normal rat kidney cells. In addition, polyclonal antibodies specific for TGF-beta or INF-gamma do not inhibit MECIF activity. MECIF preparations show low levels of TNF-alpha, insufficient to promote the observed growth inhibitory effect. MECIF activity on human endothelial cells was found to be dose dependent and reversible. MECIF also appeared to be target cell selective in that it did not significantly alter the growth of human smooth muscle cells or skin fibroblasts. These data suggest that monocyte-derived factors may play a key role in inhibiting endothelial cell proliferation.     

c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase-inactive Abl-PP-K(-) (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G(1) block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K(-) was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyrosine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis.     

Endothelial proteoglycans inhibit bFGF binding and mitogenesis

Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG-A and PG-B. The larger sized fraction (PG-A) had greater inhibitory activity than did PG-B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG-A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG-B. Both proteoglycan fractions contained perlecan-like core proteins; however, PG-A contained an additional core protein (approximately 190 kDa) that was not observed in PG-B. Both proteoglycan fractions bound bFGF directly, and PG-A bound a significantly greater relative amount of bFGF than did PG-B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF-induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis. J. Cell. Physiol. 172:209–220, 1997. © 1997 Wiley-Liss, Inc.     

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.     

Retinoic acid blocks potassium channels in human lymphocytes

Using the whole-cell variation of the patch-clamp technique, we have determined that retinoic acid, an active metabolite of natural vitamin A that possesses potent immunomodulating activity, reduces the K+ current in human T lymphocytes and natural killer cells in a dose-dependent manner: acute treatment with 5 X 10(-5) M caused over a 70% reduction while concentrations less than 1 X 10(-5) M caused less than 30% inhibition. Natural killer activity and T cell mitogenesis was inhibited by RA at concentrations that reduced the K+ conductance and correlated with the ability of a variety of classical ion-channel blockers to inhibit the functional activity of these cells. Thus, the reported inhibitory effects on natural killer activity and T cell mitogenesis by high concentrations of retinoic acid can be explained by its effect on the K channel.