Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetate in human skin fibroblasts

Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Antithrombin III stimulates prostacyclin production by cultured aortic endothelial cells

Prostacyclin (PGI2) is a potent vasodilator and an inhibitor of platelet aggregation. We found that antithrombin III (AT III), an anticoagulant present in circulating blood, stimulated PGI2 production by cultured bovine aortic endothelial cells in a dose- and time-dependent manner. The stimulation of PGI2 production by AT III was observed at physiological concentrations and was inhibited by the addition of anti-AT III antiserum and heparin. These results suggest that AT III may stimulate PGI2 production by binding to heparin-like molecules on the endothelial cell membrane.     

Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells

We have examined the effect of shear stress on the production of endothelin by cultured porcine endothelial cells. Low shear stress stimulated the expression of endothelin mRNA in polygonal endothelial cells with a peak time of 2 to 4 hours and also increased the release of immunoreactive endothelin into the culture medium. The expression of endothelin mRNA in the ellipsoidal endothelial cells under higher shear stress was not different from that of the control level. Our results suggest a possible role for hemodynamic shear stress in the regulation of endothelin production in vascular endothelial cells.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.     

Homocysteine decreases endothelin-1 production by cultured human endothelial cells.

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial-cell functionality. In-vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose-dependent decrease in both the secretion of the vasoconstrictor agent endothelin-1 (ET-1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol.L(-1)) and pharmacological noncytotoxic (1.0 and 2.0 mmol.L(-1)) concentrations. Mean percentage variation from control for ET-1 production was -36. 2 +/- 18.9% for 0.5 mmol.L(-1) homocysteine and -41.5 +/- 26.8% for 1.0 mmol.L(-1) homocysteine, after incubation for 8 h. Mean percentage variation from control for steady-state mRNA was -17.3 +/- 7.1% for 0.5 mmol.L(-1) homocysteine and -46.0 +/- 10.1 for 1.0 mmol.L(-1) homocysteine, after an incubation time of 2 h. ET-1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co-incubation of cells with homocysteine and the sulfhydryl inhibitor N-ethylmaleimide prevented the effect of homocysteine on ET-1 production, confirming a sulfhydryl-dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium-dependent vasodilation by primary inhibition of ET-1 production.     

Histamine stimulates prostacyclin synthesis in cultured human umbilical vein endothelial cells

Human venous endothelial cells synthesize prostacyclin (PGI₂) in response to treatment with histamine. The amount of PGI₂ produced is proportional to the histamine concentration over the range of 10^?7 to 10^?5 M, with a maximal response at 2–5 × 10^?6 M. PGI₂ synthesis occurs as a burst lasting less than 3 minutes after histamine addition. The H1 histamine receptor antagonist pyrilamine causes an 87% inhibition of PGI₂ synthesis, whereas the H2 antagonist cimetidine gives no significant inhibition, suggesting that PGI₂ synthesis in response to histamine is mediated by an H1 receptor.     

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.     

Transforming growth factor alpha stimulates prostacyclin production by cultured human vascular endothelial cells more potently than epidermal growth factor

Transforming growth factor alpha (TGF alpha) induces dose- and time-dependent stimulation of prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells. The lowest stimulatory concentration of TGF alpha was 0.1 ng/ml and the maximal response, a 2.7-fold rise, was obtained with 10 ng/ml. The stimulation, which lasted at least 24 h, was blocked by cycloheximide and by indomethacin. TGF alpha induced PGI2 production at 10-100 times lower concentrations than did epidermal growth factor (EGF), although in stimulating endothelial cell growth the two factors were equipotent. This is the first demonstration that TGF alpha enhances PGI2 production by human cells. Moreover, this is the first evidence that it acts as both an agonist (growth) and a superagonist (PGI2 production) of EGF in the same cell type. I suggest that this phenomenon may be involved with the angiogenic activity of TGF alpha.     

Human serum stimulates endothelin-1 synthesis more potently than prostacyclin production by cultured vascular endothelial cells

Human serum stimulated the synthesis of a vasoconstrictive peptide, endothelin-1 (ET-1), and a vasodilatory prostanoid, prostacyclin (PGI2), by cultured human umbilical vein endothelial cells in a concentration- and time-dependent manner. Incubation in 20% concentration of the serum for 24 h stimulated ET-1 synthesis almost six-fold while PGI2 production increased two-fold. In addition, a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibited the serum-induced ET-1 production and stimulated PGI2 synthesis in a concentration- and time-dependent manner. Our results suggest that human serum derived factor(s) stimulate the production of vasoconstrictive ET-1 more potently than the synthesis of vasodilatory PGI2 by human vascular endothelial cells and that the production of these agents is differentially regulated by PMA.     

Effect of human plasma lipoproteins on prostacyclin production by cultured endothelial cells

Prostacyclin (PGI2) production by bovine aortic or human umbilical vein endothelial cells increased when either human high density lipoproteins3 (HDL3) or low density lipoproteins (LDL) were added to a serum-free culture medium. At low concentrations and short incubation times, HDL3 produced more PGI2 than LDL, but LDL was just as effective as HDL3 in 18-hr incubations with high concentrations of lipoproteins. Neither lipoprotein was toxic to the cultures as assessed by [3H]leucine incorporation into cell protein. The stimulatory effect of HDL3 and LDL on PGI2 production decreased as growing cultures became confluent. Incubation with lipoproteins neither enhanced arachidonic acid release nor increased PGI2 formation when the cells were stimulated subsequently with ionophore A23187, indicating that the lipoproteins do not affect the intracellular processes involved in PGI2 production. The addition of albumin reduced the amount of PGI2 formation elicited by HDL3 or LDL. As compared with albumin-bound arachidonic acid, from 6- to 13-fold less PGI2 was produced during incubation with the lipoproteins. Furthermore, the amount of PGI2 formation elicited by the lipoproteins in 18 hr was 4-fold less than that produced during incubation with a fatty acid mixture containing only 5% arachidonic acid, and 3-fold less than when the cells were stimulated with the ionophore A23187 for 20 min. Taken together, our results indicate that human HDL and LDL contribute to endothelial PGI2 production only in a modest way and suggest that this process is not specific for either of these two plasma lipoproteins. In view of the greater participation of albumin-bound arachidonic acid in PGI2 production, plasma lipoproteins may not play as important a role in endothelial prostaglandin formation as has been suggested.     

Sphingosylphosphorylcholine stimulates CCL2 production from human umbilical vein endothelial cells

Sphingosylphosphorylcholine (SPC) is a component of high-density lipoprotein particles. We investigated the functional role of SPC in HUVECs. SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein-dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. Inhibition of p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation, but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3-, NF-κB-, and AP-1-mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Our results suggest that SPC may contribute to atherosclerosis; therefore, SPC and its unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.     

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.     

Simulated hypogravity stimulates cell spreading and wound healing in cultured human vascular endothelial cells

It is well known that endothelial cells (EC) are highly sensitive to mechanical influences such as hemodynamic conditions or pulsatile stretch. However, it is still unknown, how endothelium responds to the changed gravity. The results of some studies suggest that cellular elements of vascular wall and, particularly, endothelium, may directly participate in development of physiological responces to microgravity. On our suggestion, this is extremely attractive since vascular endothelium is one of the main regulators of vascular tone (via its interaction with vascular smooth muscle cells) and, consequently, can play not last role in maintaining of normal cardiovascular system operation in microgravity. On the other hand, the endothelium itself may be regarded as a widely dispersed organ of approximately 1.5 kg in weight (in the adult human organism). Finally, endothelium is not just a passive barrier between vascular wall and circulating blood but synthesizes, metabolizes, and releases a substances which act on adjacent cell systems or distant cell structures. The main aims of this study were: 1) the development of experimental model, allowing to study functional parameters of human endothelial cells in hypogravity conditions in vitro; 2) the verification of endothelial sensitivity to gravitational micro-environment.     

Estradiol reduces F2alpha-isoprostane production in cultured human endothelial cells

Free radical-generated F(2alpha)-isoprostanes are a group of compounds with vasoconstrictor properties. To investigate whether estradiol exerts antioxidant actions modifying F(2alpha)-isoprostane production, cultured human umbilical vein endothelial cells were exposed to estradiol and other compounds and F(2alpha)-isoprostanes were measured in culture medium. Exposure to 1 and 10 nM estradiol for 24 h reduced F(2alpha)-isoprostane production by 36 and 49%, respectively (P < 0.001 vs. control). Exposure to antiestrogens alone (ICI-182780 or EM-652) slightly reduced F(2alpha)-isoprostanes (P < 0.05 vs. control), but much less than exposure to estradiol (P < 0.05). ICI-182780 reversed the estradiol-induced reduction of F(2alpha)-isoprostane concentration (P < 0.05). Along with time-course analysis, these results suggest that estradiol effects were mediated through estrogen receptor-dependent and -independent mechanisms. Progestogens alone (progesterone or medroxyprogesterone acetate) did not modify F(2alpha)-isoprostane production at any of the tested concentrations (1, 10, and 100 nM). Progesterone completely reversed estradiol-induced reduction of F(2alpha)-isoprostane production (P < 0.05 vs. control and estradiol), but medroxyprogesterone acetate did not (P < 0.05 vs. control).