Bleomycin upregulates gene expression of angiotensin-converting enzyme via mitogen-activated protein kinase and early growth response 1 transcription factor.

Pulmonary fibrosis is a progressive disorder characterized by the loss of alveolar architecture through epithelial and endothelial cell apoptosis and fibroblast proliferation. Recent studies showed that angiotensin-converting enzyme (ACE) activity is increased in fibrotic tissues, and ACE inhibitors administered in vivo ameliorate fibrosis, suggesting that ACE may play a critical role. However, the regulation of ACE expression is not well understood. In the present study, we demonstrate that bleomycin, a chemotherapeutic agent which induces pulmonary fibrosis in animals and humans, increases gene expression of ACE. Treatment of primary bovine pulmonary artery endothelial cells with 0.1 to 1.0 microg/ml bleomycin increased ACE enzymatic activity and ACE mRNA, as monitored by hippuryl-L-histidyl-L-leucine assay and competitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Luciferase reporter constructs showed that upregulation of ACE transcription by bleomycin is mediated through element(s) in the 97-bp ACE promoter. Bleomycin activated p42/p44 mitogen-activated protein kinase (MAPK) and induced nuclear translocation and activation of the early growth response (Egr)-1 transcription factor, a factor previously shown to positively regulate ACE expression. The MAPK kinase1/2 (MEK1/2) inhibitor U0126 blocked MAPK and Egr-1 activation by bleomycin, suggesting that Egr-1 activation is MAPK dependent. These data provide the first evidence that bleomycin activates ACE gene expression through the MAPK pathway and Egr-1.

The role of endothelin-1 in strain-related susceptibility to develop hypoxic pulmonary hypertension in rats.

The Hilltop (H) strain compared to the Madison (M) strain of Sprague-Dawley rats develops severe pulmonary hypertension in response to chronic hypoxia. We tested the hypothesis that endothelin-1 (ET-1) contributes to these strain-related differences. Plasma ET-1 content was not modified by chronic hypoxia in either strain. The lung ET-1 peptide and preproET-1 mRNA content were significantly increased to the same magnitude in both strains at 2 and 3 weeks of hypoxia. The ET(A) receptor mRNA increased more at 3 weeks of hypoxia in the lungs of H rats than in M rats, but not at other time points. The ET(B) receptor mRNA was not modified by hypoxia in either strain. After 3 days of normoxic recovery following 2 weeks of hypoxia, ET-1 protein and mRNA levels decreased to baseline levels in both rat strains. We conclude that ET-1 does not contribute to the development of cardiopulmonary differences between the H and M strains in response to hypoxia.

Reduction of endothelial cell related TGFbeta activity by thiols.

Transforming growth factor beta (TGFbeta) may play an important role in diseases characterized by pulmonary fibrosis. We have previously demonstrated that thiols inhibit the pro-oxidant effects of TGFbeta1 in bovine pulmonary artery endothelial cells (BPAEC). To help define the mechanism of this observation we have examined the effect of reduced (GSH) and oxidized (GSSG) glutathione, N-acetyl cysteine (NAC) and cysteine (CYS) on the biological activity of a) TGFbeta released by bovine pulmonary artery endothelial cells (BPAEC) into culture medium, and b) commercially available porcine platelet TGFbeta1. The biological activity of TGFbeta (following activation) released into the medium from cultured BPAEC was significantly reduced when the cells were cultured in the presence of 10 mM GSH or 10 mM NAC for 24 h (10 mM GSH: 85.7 +/- 50 pg/ml/10(6) cells and 10 mM NAC: 127.3 +/- 35 pg/ml/10(6) cells, compared with control: 541 +/- 8.9 pg/ml/10(6) cells; p < 0.05). Thiols (10 mM GSH, 10 mM NAC and 5 mM cysteine), added directly to cell-free conditioned medium or to a commercially available preparation of porcine platelet TGFbeta1 for 6-24 h had a similar inhibitory effect on the biological activity of TGFbeta and altered the structure of porcine platelet TGFbeta1 as determined by mass spectroscopy. These thiols failed to reduce the expression of TGFbeta mRNA in BPAEC as measured by a competitive polymerase chain reaction assay. Incubating endothelial cells or cell-free conditioned medium with GSSG did not alter the biological activity of TGFbeta. Lower doses of thiols (0.1-1 mM), that we have shown inhibit the antiproliferative and pro-oxidant effects of exogenous TGFbeta1 on BPAEC, had no direct effect on TGFbeta bioactivity. In summary, thiols are capable of reducing the effects of TGFbeta in biological systems through a direct effect on the TGFbeta molecule. However, this action appears to be dose-dependent, and at low doses (0.1-1 mM) thiols may also inhibit the actions of exogenous TGFbeta1 in cell culture through a mechanism involving the cellular redox status.

Superoxide scavenging effect of Ginkgo biloba extract on serotonin-induced mitogenesis.

We have reported previously that serotonin (5-HT) stimulates the mitogenesis of bovine pulmonary artery smooth muscle cells (SMCs) through active transport of 5-HT and cellular signaling that includes elevation of superoxide (O2.-) and enhancement of protein tyrosine phosphorylation. Ginkgo biloba extract 501 (EGb 501), which has been demonstrated to act as an antioxidant, was found to block both the elevated O2.- and the proliferative and hypertrophic influences of 5-HT on SMCs, but not to directly inhibit the associated activation of NAD(P)H oxidase or the stimulation of phosphorylation of GTPase-activating protein (GAP). A similar effect of Ginkgo biloba extract 501 occurred on Chinese hamster lung fibroblasts (CCL-39), where 5-HT receptor, as opposed to transporter, action has been associated with mitogenesis. We conclude from these studies that Ginkgo biloba extract 501 quenches O2.- formation by 5-HT, thereby blocking its mitogenic effect. Stimulation of protein tyrosine phosphorylation of GAP by 5-HT appears to precede the elevation of O2.-.

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury.

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1beta (100 microg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1beta or hypoxia independently produced a 2-fold elevation (p < 0. 05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1beta and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1beta alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1beta, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1beta, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.

Determination of xanthine dehydrogenase mRNA by a reverse transcription-coupled competitive quantitative polymerase chain reaction assay: regulation in rat endothelial cells by hypoxia and hyperoxia.

The enzyme system xanthine dehydrogenase (XD):xanthine oxidase, which generates the superoxide anion as a by-product of action on endogenous substrates, is believed to play a role in mediating pathophysiological changes through its contribution to total superoxide production. To aid with analysis of factors that regulate XD, we have developed a reverse transcription (RT)-coupled competitive quantitative polymerase chain reaction (PCR) assay which enables XD mRNA to be determined from small amounts of cultured cells where constitutive XD levels are low. A homologous insertion mutant of wild-type XD cDNA was prepared and used as an internal standard to normalize intersample PCR efficiency differences. XD mRNA levels determined by RT-PCR also were normalized to tubulin mRNA to compensate for RT differences and loading effects among samples. We report that XD mRNA levels, determined by RT-PCR, were increased twofold in hypoxic (3% oxygen) rat pulmonary microvascular endothelial cells relative to normoxic controls (20% oxygen). Conversely, XD mRNA was decreased threefold within 24 h under hyperoxic (95% oxygen) conditions. These data support the hypothesis that XD is regulated by oxygen tension in the pulmonary vasculature.

Quantification of polymerase chain reaction products: enzyme immunoassay based systems for digoxigenin- and biotin-labelled products that quantify either total or specific amplicons.

Enzyme immunoassays were developed for quantification of polymerase chain reaction (PCR) products, referred to as amplicons. Amplicons were dual labelled simultaneously by enzymatic incorporation of digoxigenin and biotin during PCR. For total amplicon quantification, Microfluor B polystyrene wells, compatible with chemiluminescent detection, were coated with streptavidin. Dual labelled amplicons were bound, treated with anti-digoxigenin antibody conjugated to alkaline phosphatase to complete the two-site sandwich immunoassay configuration, and detected by the chemiluminescence generated upon hydrolysis of a phosphate substituted dioxetane substrate, AMPPD. For specific amplicon quantification, the Microfluor B wells were coated with an unlabelled DNA probe complementary to the labelled amplicon target. Subsequent steps were performed as described above. This assay detects 2 pg of specifically amplified DNA. Chemiluminescent detection provides a linear range of four orders of magnitude for amplicon quantification. The non-radioactive labels are safe and stable. PCR as described here obviates the need for labelled primers and constitutes the initial report of concurrent dual non-radioactive labelling of DNA by a DNA polymerase.

A competitive quantitative polymerase chain reaction assay for bovine transforming growth factor-B1 mRNA.

We have developed a flexible reverse transcription (RT) coupled quantitative polymerase chain reaction (PCR) assay for transforming growth factor beta-1 (TGF-b) mRNA. A deletion mutant cDNA internal standard was prepared from the wild type cDNA and used to normalize intersample PCR efficiency differences. The assay is compatible with samples from cow and other species. Using RT-PCR, we determined that TGF-b mRNA in bovine pulmonary artery endothelial cells is increased by TGF-b 7.5-fold within 6h and remains 4-fold above baseline after 12h. In addition, unlike TGF-b bioactivity, mRNA levels in endothelial cells are not decreased upon exposure of the cells to either glutathione (reduced or oxidized), cysteine, or N-acetylcysteine for 24h.

Effect of nitric oxide and cell redox status on the regulation of endothelial cell xanthine dehydrogenase.

We have previously reported that endothelial cell (EC) xanthine dehydrogenase/xanthine oxidase (XD/XO) activity correlates inversely with the O2 tension to which the cells are exposed. Whether this effect is related to the production of reactive O2 species is unclear. We exposed bovine pulmonary artery EC to various conditions that altered the redox status of the cells: 1) hypoxia (3% O2) and normoxia (20% O2); 2) menadione (MEN), known to generate O2 radicals; 3) catalase (CAT) and reduced glutathione (GSH), which detoxify H2O2; and 4) various NO-generating systems. Changes in intracellular XO and XO + XD activities were correlated with rates of extracellular H2O2 release from the same cells. Conditions that decreased extracellular H2O2 release (hypoxia, CAT, and GSH) produced significant and parallel increases in intracellular XO and XO + XD activities in a time-dependent fashion. MEN treatment increased extracellular release of H2O2 and subsequently reduced intracellular XO and XO + XD activities. NO-generating agents did not change extracellular release of H2O2 but significantly reduced XO and XO + XD activities. The latter effect was prevented by reduced hemoglobin. Scavengers of hydroxyl radicals reversed the inhibition of XO and XO + XD activities produced by MEN but not that produced by NO. While NO significantly inhibited XD/XO activity from rat epididymal fat pad, it did not affect XD/XO mRNA expression in these cells. We conclude that intracellular XD/XO activity is sensitive to changes in oxidant-generating and protective systems. Inhibition of XD/XO activity by NO may be mediated through direct binding of NO to the enzyme iron-sulfur moiety or to its sulfhydryl groups.(ABSTRACT TRUNCATED AT 250 WORDS)

A competitive deletion mutant quantitative PCR assay for angiotensin-converting enzyme mRNA in smooth muscle cells.

To quantify angiotensin-converting enzyme (ACE) mRNA, we have developed a reverse transcription (RT)-coupled competitive PCR (RT-PCR) assay with a deletion mutant internal standard. The RT-PCR detects ACE mRNA from both human and bovine sources. ACE mRNA was detected in total RNA from cultured human saphenous vein smooth muscle cells (HuSV-SMCs) and from bovine pulmonary artery (BPA) SMCs. BPA-SMC expressed ninefold less ACE mRNA than BPA endothelial cells, and threefold less than HuSV-SMCs. Apparent amounts of ACE mRNA were 118,350 +/- 2,300 copies in HuSV-SMCs and 42,200 +/- 11,300 copies in BPA-SMCs per microgram of total cell RNA. The accuracy of the absolute values is subject to the limitations of the assumptions used to calculate them. These data support the hypothesis that components of the renin-angiotensin system are transcribed by SMCs.

Relative quantification of angiotensin-converting enzyme mRNA in human smooth muscle cells, monocytes, and lymphocytes by the polymerase chain reaction.

A method was developed for relative quantification of angiotensin-converting enzyme (ACE) mRNA in as few as 100 cells. After reverse transcription of total RNA to cDNA, multiplexed polymerase chain reaction with two sets of primers amplified ACE cDNA and that of an internal standard glyceraldehyde phosphate dehydrogenase (GAPDH) simultaneously. By adjusting primer pair concentrations, both ACE and GAPDH were amplified with constant efficiencies. Macrophage-like U937 histiocytic lymphoma cells expressed ACE mRNA constitutively. Freshly isolated human monocytes did not express ACE mRNA initially, but after 4 days in culture had 8% of the amount found in U937 cells. After phorbol ester stimulation, monocytes transcribed ACE at levels comparable to U937 cells. Human smooth muscle cells had ninefold more ACE mRNA than 4-day monocytes, but 30% less than U937 cells. In contrast, a mixed population of lymphocytes was devoid of ACE mRNA.

Angiotensin converting enzyme (kininase II) mRNA production and enzymatic activity in human peripheral blood monocytes are induced by GM-CSF but not by other cytokines.

Peripheral blood monocytes (PBM) do not possess angiotensin converting enzyme (ACE) activity in the inactive state. However, measurable PBM ACE activity is found in patients with certain inflammatory diseases. We have examined the effect of cytokines likely to be present during granulomatous inflammation on the regulation of ACE mRNA in PBM. The presence of ACE mRNA in human PBM cultured in vitro with various cytokines for up to 6 days was analyzed using polymerase chain reaction. PBM not exposed to cytokines did not express ACE mRNA, while incubation of PBM with recombinant human GM-CSF resulted in high levels of ACE mRNA expression after 72 h of cell culture, which persisted through day six. Increased ACE mRNA expression occurred concomitantly with phenotypic changes in cell size and shape consistent with cell activation. A 5-fold increase in ACE enzymatic activity also occurred. Incubation of PBM with all other cytokines tested failed to induce ACE mRNA expression. Alveolar macrophages expressed ACE mRNA immediately following their isolation, but mRNA expression decreased markedly during a 24-h period of incubation and was only partially reversed with exogenous GM-CSF. We conclude that GM-CSF enhances ACE mRNA levels in human PBM, but not in alveolar macrophages.

Regulation of endothelial cell xanthine dehydrogenase xanthine oxidase gene expression by oxygen tension.

Recent studies have demonstrated that xanthine dehydrogenase/xanthine oxidase (XD/XO) activities of bovine endothelial cells (EC) are inversely regulated by O2 tensions to which the cells are exposed. We have confirmed these reports and extended the observation to a variety of cells from other sources. All EC that had detectable XD/XO activity demonstrated the greatest activity at the lowest O2 level. Bovine pulmonary artery smooth muscle cells showed XD/XO activity only under hypoxic conditions. The ratio of XO to XO+XD did not change significantly under various O2 concentrations for all cell types tested. Treatment of bovine pulmonary artery and rat epididymal fat pad EC with actinomycin D (1 microgram/ml), an inhibitor of transcription, suppressed XO and XO+XD activities in cells exposed both to 20 and 3% O2. High-dose cycloheximide (5 micrograms/ml), an inhibitor of translation, also reduced XO and XO+XD activities in these cells, whereas low-dose cycloheximide (0.5 microgram/ml) enhanced the stimulatory effect of hypoxia on XO+XD activity. We developed a digoxigenin-labeled probe that recognizes and hybridizes to rat XD cDNA and used it to examine the effect of O2 concentration on XD/XO mRNA expression of rat epididymal fat pad EC. XD/XO mRNA concentration was increased in cells exposed to hypoxia and decreased in cells exposed to hyperoxia compared with normoxic cells. The increase in mRNA concentration resulting from exposure to hypoxia was enhanced by cycloheximide. There was no change in XD/XO mRNA stability in cells exposed to hypoxia compared with normoxia. We conclude that the regulation of XD/XO by oxygen tension most likely occurs at the transcriptional level.

Regulation of serotonin-induced DNA synthesis of bovine pulmonary artery smooth muscle cells.

We have previously reported that serotonin [5-hydroxytryptamine (5-HT)] stimulates DNA synthesis of bovine pulmonary artery smooth muscle cells (SMC) by its high-affinity uptake. Uptake inhibitors, but not selective 5-HT receptor antagonists, prevented the stimulatory effect (S.-L. Lee and B. L. Fanburg. J. Cell. Physiol. 150:396-405, 1992). We have now further evaluated the mechanism by which 5-HT enhances SMC DNA synthesis. Although some serotonergic agonists mimicked this stimulation, selective 5-HT receptor agonists produced no or only minor and variable stimulatory effects. The action of 5-HT was not inhibited by inhibitors of phospholipases C and A2, the protein kinase C (PKC) inhibitors dihydrosphingosine and 1-(-isoquinolinylsulfonyl)-2 methylpiperazine (H-7), or down-regulation of PKC with phorbol 12,13-dibutyrate. Staurosporine, a reputed PKC and tyrosine kinase (TK) inhibitor, and genistein, a selective TK inhibitor, reversed the stimulatory effect of 5-HT in a dose-dependent manner. Before stimulation of thymidine incorporation into cellular DNA, 5-HT elevated c-myc and actin mRNAs. Imipramine, fluoxetine, staurosporine, and cholera toxin inhibited the stimulations of both DNA synthesis and c-myc and actin mRNA expressions by 5-HT. Thus the data support a concept that 5-HT-induced thymidine incorporation by SMC involves membrane transport of 5-HT that initiates tyrosine phosphorylation.

Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture.

Serotonin [5-hydroxytryptamine (5-HT)] has a dual effect on bovine pulmonary artery smooth muscle cells (SMC) in culture (S.-L. Lee, W. W. Wang, B. J. Moore, and B. L. Fanburg. Circ. Res. 68: 1362-1368, 1991.). Cellular internalization of 5-HT stimulates DNA synthesis and cellular proliferation, whereas the action of 5-HT on a cell surface receptor elevates adenosine 3',5'-cyclic monophosphate and inhibits proliferation. The present study shows that 5-HT causes proliferation of both pulmonary artery and aortic SMC but not of endothelial cells or fibroblasts. Furthermore, c-myc and alpha- and beta-actin gene expressions of pulmonary artery SMC were elevated after 2-h incubation with 5-HT, before stimulation of [3H]thymidine incorporation. Actinomycin D (0.05 micrograms/ml) but not cycloheximide (1 microgram/ml) inhibited the gene expression produced by 5-HT. Growth-arrested SMC progressed from a G0 quiescent state through a normal cell cycle when subjected to 5-HT, 5-HT plus 25 ng/ml insulin-like growth factor, or 5-HT plus 0.5 ng/ml fibroblast growth factor. Cell number increased by 20-40% at 40 h and 50-140% at 7 days. Protein content of cells treated with 5-HT was elevated by 20-40% at 7 days, whereas the rate of protein synthesis, measured by [35S]methionine incorporation, increased by 50-70% at 24 h. In the presence of 1 microM 5-HT, cells enlarged by 70 and 100-200% at 40 h and 7 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cu,Zn superoxide dismutase in vascular cells: changes during cell cycling and exposure to hyperoxia.

Conditions that generate reactive oxygen species elevate Cu,Zn superoxidase dismutase (SOD) in endothelial cells (EC) concomitant with decreased cellular proliferation. The current studies were undertaken with both vascular EC and smooth muscle cells (SMC) to compare the influences of cellular proliferation with those of hyperoxia on induction of Cu,Zn SOD. To assess cell cycling alone, EC and SMC were growth arrested, then released from arrest. Cell cycling was monitored by [3H]thymidine incorporation, counts, and flow cytometry. SOD catalytic activity was measured spectrophotometrically and SOD protein by enzyme-linked immunosorbent assay. A digoxigenin-labeled probe was used to quantify SOD mRNA by Northern analysis. EC reached the S phase of the cell cycle in 18 h and completed one cycle in 24-30 h, whereas SMC took 24-30 h to reach the S phase and 48 h to complete one cycle. Cu,Zn SOD mRNA for both EC and SMC was very low during the Go/G1 phase, peaked during the S phase, and then reverted to lower values as cells progressed through their cycles. Cu,Zn SOD activity and immunoprotein content showed corresponding changes to those of mRNA. Exposure to hyperoxia (95% O2) delayed the entry of released cells into the S phase of the cell cycle and blocked the cells in the S or G2 phase, but induced Cu,Zn SOD mRNA before the S phase and caused persistence of elevation of Cu,Zn SOD mRNA as cells progressed through their cycles. Exposure to hyperoxia also induced Cu,Zn SOD mRNA in growth-arrested cells within 24-48 h. Thus our studies support roles for both cells cycle dependency and reactive oxygen species in the induction of Cu,Zn SOD.

Stimulation of bovine pulmonary artery endothelial cell ACE by dexamethasone: involvement of steroid receptors.

After exposure of bovine pulmonary artery endothelial cells in culture to 1 microM dexamethasone for 24-48 h, angiotensin-converting enzyme (ACE) activity of these cells was elevated severalfold. The increase in ACE activity was preceded by an increase in ACE mRNA, which could be detected after treatment of cells with dexamethasone for 4 h. When the increase in ACE mRNA produced by dexamethasone at 4 h was blocked by alpha-amanitin, an RNA polymerase II inhibitor, the increase in ACE activity detected at 48 h was inhibited. RU 38486, a steroid receptor antagonist, inhibited the elevation of both ACE activity and mRNA produced by dexamethasone. Among other steroids tested, only hydrocortisone, aldosterone and corticosterone-21-acetate had a stimulatory effect on ACE activity. RU 38486 effectively blocked the elevation in ACE activity produced by both aldosterone and dexamethasone, but had no effect on the elevation of ACE activity produced by other agents (3-isobutyl-1-methylxanthine, A23187, and dibutyryl adenosine 3',5' cyclic monophosphate). From these data we conclude that dexamethasone and certain other steroids with an hydroxyl group in the 11th carbon position regulate ACE gene expression of bovine endothelial cells at the transcriptional level via a steroid receptor-mediated mechanism.

Inhibition of pulmonary artery smooth muscle cell growth by hypoxanthine, xanthine, and uric acid.

We have previously reported that medium conditioned by hypoxic pulmonary artery endothelial cells (ECCM) contains a factor of small molecular weight that inhibits the growth of pulmonary artery smooth muscle cells (SMC). We postulated that this factor might be a breakdown product of ATP and, therefore, measured the levels of hypoxanthine/xanthine (HX/X) and uric acid (UA) in ECCM and cell lysates from endothelial cells (EC) exposed to hypoxia and normoxia. Although hypoxic and normoxic cell lysates contained no UA and an equal amount of HX/X (2.9 +/- 0.3 and 2.9 +/- 0.5 microM, respectively), there was a 5-fold increase in the amount of HX/X present in hypoxic compared with normoxic ECCM (3.4 +/- 0.3 versus 0.6 +/- 0.4 microM, respectively; P less than 0.001) but no difference in UA levels (5 +/- 2 versus 5 +/- 1 microM, respectively). In separate experiments, we examined the effects of exogenous HX, X, and UA (doses ranging from 0.1 to 100 microM) on the proliferation of pulmonary and aortic SMC and pulmonary artery EC. Our results indicate that HX, X, and UA inhibit the proliferation of SMC in a dose-dependent manner without causing injury to the cells. The proliferation of EC, on the other hand, was not affected by UA and was significantly inhibited by HX and X only at doses of 100 microM. In conclusion, we have found that significant amounts of HX/X accumulate in hypoxic ECCM and that HX, X, and UA inhibit the proliferation of SMC. The relevance of these findings to conditions where hypoxia prevails is discussed.