Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype

We previously reported that adenosine A2B receptor activation stimulates angiogenesis. Because hypoxia is a potent stimulus for the release of both adenosine and angiogenic factors, we tested the hypothesis that hypoxia alters the expression of adenosine receptors toward an "angiogenic" phenotype. We used human umbilical vein endothelial cells (HUVECs) and bronchial smooth muscle cells (BSMCs) because, under normoxic conditions, adenosine does not release vascular endothelial growth factor (VEGF). HUVECs expressed a characteristic A2A phenotype (the selective A2A agonist CGS21680 was as potent as the nonselective agonist 5'-N-ethylcarboxamidoadenosine [NECA] in generating cAMP). Hypoxia (4.6% O2, 3 hours) decreased A2A mRNA from 1.56+/-0.3% to 0.16+/-0.01% of beta-actin expression but increased A2B mRNA from 0.08+/-0.01% to 0.27+/-0.05%. Consistent with changes in receptor expression, CGS21680 failed to increase cAMP in hypoxic HUVECs, whereas NECA remained active (A2B phenotype), and NECA increased VEGF release from 9.5+/-1.0 to 14.2+/-1.2 pg/mL (P<0.05), indicating that increased A2B receptors were functionally coupled to upregulation of VEGF. Hypoxia had similar effects on BSMCs, increasing A2B mRNA by 2.4+/-0.3-fold, from 0.42+/-0.04% to 1.00+/-0.13% of beta-actin. Whereas NECA had no effect on VEGF release in normoxic BSMCs, it increased VEGF release in hypoxic BSMCs, from 74.6+/-9.6 to 188.3+/-16.7 pg/mL (P<0.01), and a selective A2B antagonist, CVT-6694, inhibited this increase. A2B receptors activated a VEGF reporter made unresponsive to hypoxia by mutating its hypoxia-inducible factor-1 (HIF-1) binding element, indicating a mechanism independent of HIF-1. In conclusion, hypoxia modulates the expression of adenosine receptors in human endothelial and smooth muscle cells toward an A2B"angiogenic" phenotype.     

Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-alpha (TNF-alpha) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-alpha was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the alpha(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against alpha(v)beta(3)- and alpha(v)beta(5)-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (Blood. 2000;96:2775-2783)     

Constitutive phosphodiesterase activity restricts spontaneous beating rate of cardiac pacemaker cells by suppressing local Ca2+ releases

We hypothesized that activation of angiogenesis by chronic hypoxia may affect vascular resistance and, subsequently, blood pressure levels in spontaneously hypertensive rats (SHRs). Five-week-old prehypertensive SHRs and age-matched normotensive Wistar-Kyoto (WKY) rats (n=8 per group) were maintained under normobaric normoxic or hypoxic (10% O(2)) conditions for 8 weeks. Three weeks later, the systolic blood pressure was lower by 26% in hypoxic SHRs compared to normoxic SHRs (P<0.05) and remained at the normoxic WKY level. Total peripheral vascular resistance, calculated as the mean arterial pressure/cardiac output (assessed by ultrasound imaging and Doppler), was 30% lower in hypoxic than in normoxic SHRs (P<0.001) and returned to WKY levels. Interestingly, chronic hypoxia also significantly reduced systolic blood pressure in adult 12-week-old SHRs with established hypertension; blood pressure was normalized (versus normoxic WKY rats) after 4 weeks of hypoxia. Changes in hemodynamic parameters were associated with activation of proangiogenic pathways. Protein levels of vascular endothelial growth factor (VEGF)-A in the skeletal muscles were increased by 2.2-fold in hypoxic compared to normoxic SHRs (P<0.001). At the end of the hypoxic period, capillary density in the quadriceps muscle was 1.2-fold higher in hypoxic than in normoxic SHRs (P<0.001). Myocardial capillary density and VEGF-A protein contents were also 1.2- and 2.1-fold higher in hypoxic compared to normoxic SHRs (P<0.001 and P<0.05, respectively). Moreover, treatment with neutralizing VEGF-A antibody abrogated the hypoxia-induced angiogenesis and subsequently worsened arterial hypertension. Therefore, our results suggest that chronic normobaric hypoxia (1) activates VEGF-A-induced angiogenesis and thereafter (2) prevents the occurrence of hypertension in young prehypertensive SHRs and (3) normalizes blood pressure in adult SHRs with established hypertension.     

Retinal endothelial angiogenic activity: effects of hypoxia and glial (Müller) cells

OBJECTIVE: To explore the impact of retinal glial (Müller) cells on survival and neovascularization-related activities of cultured retinal endothelial cells under normoxic and hypoxic conditions. METHODS: Bovine retinal endothelial cells (BRECs) were cultured under normoxia or hypoxia (0.5% O2) either alone, together with the human Müller cell line MIO-M1, or in normoxia- or hypoxia-conditioned media of MIO-M1 cells. Cell number, proliferation, apoptotic cell death, and migration of BRECs were determined. RESULTS: Exposure of BRECs to hypoxia for 24 h decreased the number of adherent cells and the proliferation rate, but increased apoptosis and cell migration. Increased apoptosis and decreased proliferation of the BRECs occurred also in the presence of conditioned media of MIO-M1 cells. Under normoxic conditions, co-culture with MIO-M1 cells resulted in increased proliferation, but decreased apoptosis and migration rates of BRECs. Under hypoxic conditions, the Müller cells released elevated amounts of VEGF but their presence decreased proliferation, apoptosis and the migration rates of BRECs. CONCLUSIONS: Hypoxia inhibits the proliferation of retinal endothelial cells. Müller cells release soluble mediators that enhance this hypoxia-mediated effect but, under certain conditions (i.e., in co-culture), may protect retinal endothelial cells from apoptosis, thus supporting their survival. Altogether the findings indicate that the key signal necessary to trigger retinal endothelial proliferation under hypoxia remains to be determined.     

Notch-1信号通路对胰腺癌BxPC3细胞增殖和凋亡的调控

目的探讨Notch-1信号通路对胰腺癌BxPC3细胞增殖和凋亡的影响。方法体外培养胰腺癌BxPC3细胞,用小干扰RNA（small interference RNA,siRNA）的方法抑制Notch-1的表达,Western印迹方法检测Notch-1、Bcl-2及Bcl-xL蛋白表达情况;MTT法检测细胞增殖;ELISA法检测细胞凋亡。结果抑制Notch-1表达能明显抑制胰腺癌BxPC3细胞生长（P〈0．01）,显著下调Bcl-2和Bcl-xL水平,并使BxPC3细胞凋亡显著增多（P〈0．01）。结论Notch-1信号通路促进胰腺癌BxPC3细胞增殖,抑制胰腺癌细胞凋亡。     

肝细胞生长因子对糖基化终产物诱导内皮细胞凋亡的影响

目的探讨糖基化终产物对内皮细胞的凋亡作用,以及肝细胞生长因子对内皮细胞凋亡的影响。方法体外培养人脐静脉内皮细胞,予不同浓度糖基化终产物及肝细胞生长因子干预,分为实验对照组及100 mg/L、200mg/L4、00 mg/L糖基化终产物组和400 mg/L糖基化终产物 100μg/L肝细胞生长因子组,采用四甲基偶氮唑蓝比色法测定各组内皮细胞生长抑制率,通过吖啶橙荧光染色观察细胞形态学变化,流式细胞术测定Annexin V-FITC/PI双染标记的细胞凋亡率,检测肝细胞生长因子对糖基化终产物诱导内皮细胞凋亡的影响;蛋白免疫印迹法分析各组凋亡基因Bax、Bcl-2蛋白的表达及酶联反应法测定细胞凋亡蛋白酶3的活性。结果肝细胞生长因子能明显降低糖基化终产物对内皮细胞生长的抑制作用(P<0.01);糖基化终产物诱导培养的内皮细胞出现明显的凋亡形态学改变,在一定浓度范围内,内皮细胞凋亡率与糖基化终产物的浓度和作用时间呈依赖关系,肝细胞生长因子干预后可显著降低不同时间的内皮细胞凋亡率(P<0.05);肝细胞生长因子作用内皮细胞抗凋亡基因Bcl-2表达明显升高(P<0.01),而促凋亡基因Bax表达无明显变化(P>0.05);细胞凋亡蛋白酶3活性显著降低(P<0.05)。结论糖基化终产物诱导人内皮细胞凋亡,而肝细胞生长因子抑制糖基化终产物诱导的内皮细胞凋亡,其作用机制可能是上调抗凋亡基因Bcl-2水平、抑制细胞凋亡蛋白酶3的激活。     

A cellular paradigm for the failure to increase vascular endothelial growth factor in chronically hypoxic states.

BACKGROUND: Angiogenesis, or new blood vessel formation, is the physiological adaptation of a tissue to hypoxia or ischemia. However, this compensatory response to hypoxic stress in vivo is often insufficient. In many of the conditions in which the angiogenic response to tissue hypoxia is insufficient, such as chronic critical limb ischemia or myocardial hibernation, the hypoxic stress is chronic and persistent, lasting for days or even months. Vascular endothelial growth factor (VEGF) has been demonstrated in vivo and in vitro to be the principal mediator of hypoxia-induced angiogenesis. We propose that the lack of compensatory angiogenesis in response to tissue hypoxia in many clinical syndromes characterized by chronic hypoxia is due to a failure to induce VEGF appropriately. METHODS AND RESULTS: Heart or liver cells were grown under conditions of chronic hypoxia, returned to a normoxic environment, and then rechallenged with hypoxia. We found that the hypoxic induction of VEGF mRNA was markedly blunted using this algorithm. Furthermore, transient transfection studies using the VEGF promoter containing an oxygen-responsive enhancer element failed to show induction in cells pretreated by subjection to chronic hypoxia. CONCLUSIONS: Hypoxic pretreatment results in a blunting of the ability of a cell to induce VEGF in response to subsequent episodes of hypoxia. This may provide a rationale for the inadequate amount of compensatory angiogenesis seen in many chronic ischemic disorders.     

Inflammatory mediators are perpetuated in macrophages resistant to apoptosis induced by hypoxia

A hypoxic/anoxic microenvironment has been proposed to exist within a vascular lesion due to intimal or medial cell proliferation in vascular diseases. Here, we examined whether hypoxia alters macrophage function by exposing murine macrophage-like RAW 264.7 (RAW) cells to hypoxia (2% O₂). When cells were exposed to hypoxia, a significant number of RAW cells underwent apoptosis. Additionally, small subpopulations of RAW cells were resistant to hypoxia-induced apoptosis. Through repeated cycles of hypoxia exposure, hypoxia-induced apoptosis-resistant macrophages (HARMs) were selected; HARM cells demonstrate >70% resistance to hypoxia-induced apoptosis, as compared with the parental RAW cells. When heat shock protein (HSP) expression was examined after hypoxia, we observed a significant decrease in constitutive heat shock protein 70 (HSC 70) in RAW cells, but not in HARMs, as compared with the control normoxic condition (21% O₂). In contrast, the expression level of glucose-regulated protein 78 (GRP 78) in RAW and HARM cells after hypoxia treatment was not altered, suggesting that HSC 70 and not GRP 78 may play a role in protection against hypoxia-induced apoptosis. When tumor necrosis factor α (TNF-α) production was examined after hypoxic treatment, a significant increase in TNF-α production in HARM but decrease in RAW was observed, as compared with cells cultured in normoxic conditions. HARM cells also exhibit a much lower level of modified-LDL uptake than do RAW cells, suggesting that HARMs may not transform into foam cells. These results suggest that a selective population of macrophages may adapt to potentially pathological hypoxic conditions by overcoming the apoptotic signal.     

Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.     

Hypoxia induces angiogenic factors in brain microvascular endothelial cells

Hypoxia is increasingly recognized as an important contributing factor to the development of brain diseases such as Alzheimer's disease (AD). In the periphery, hypoxia is a powerful regulator of angiogenesis. However, vascular endothelial cells are remarkably heterogeneous and little is known about how brain endothelial cells respond to hypoxic challenge. The objective of this study is to characterize the effect of hypoxic challenge on the angiogenic response of cultured brain-derived microvascular endothelial cells. Brain endothelial cell cultures were initiated from isolated rat brain microvessels and subjected to hypoxia (1% O(2)) for various time periods. The results showed that hypoxia induced rapid (≤ 0.5h) expression of hypoxia-inducible factor 1α (HIF-1α) and that cell viability, assessed by MTT assay, was unaffected within the first 8h. Examination of brain endothelial cell cultures for pro- and anti-angiogenic proteins by western blot, RT-PCR and ELISA revealed that within 0.5 to 2h of hypoxia levels of vascular endothelial growth factor and endothelin-1 mRNA and protein were elevated. The expression of heme oxygenase-1 also increased but only after 8h of hypoxia. In contrast, similar hypoxia exposure evoked a decrease in endothelial nitric oxide synthase and thrombospondin-2 levels. Exposure of brain endothelial cell cultures to hypoxia resulted in a significant (p<0.001) decrease (94%) in tube length, an in vitro index of angiogenesis, compared to control cultures. The data indicate that, despite a shift toward a pro-angiogenic phenotype, hypoxia inhibited vessel formation in brain endothelial cells. These results suggest that in brain endothelial cells expression of angiogenic factors is not sufficient for the development of new vessels. Further work is needed to determine what factors/conditions prevent hypoxia-induced angiogenic changes from culminating in the formation of new brain blood vessels and what role this may play in the pathologic changes observed in AD and other diseases characterized by cerebral hypoxia.     

Hypoxia enhances Ecto-5'-Nucleotidase activity and cell surface expression in endothelial cells: role of membrane lipids

Extracellular adenosine production by the glycosyl-phosphatidyl-inositol-anchored Ecto-5'-Nucleotidase plays an important role in the defense against hypoxia, particularly in the intravascular space. The present study was designed in order to elucidate the mechanisms underlying hypoxia-induced stimulation of Ecto-5'-Nucleotidase in endothelial cells. For this purpose, aortic endothelial cells (SVARECs) were submitted to hypoxic gas mixture. Hypoxia (0% O2 for 18 hours) induced a 2-fold increase of Ecto-5'-Nucleotidase activity (Vmax 19.78+/-0.53 versus 8.82+/-1.12 nmol/mg protein per min), whereas mRNA abundance and total amount of the protein were unmodified. By contrast, hypoxia enhanced cell surface expression of Ecto-5'-Nucleotidase, as evidenced both by biotinylation and immunostaining. This effect was accompanied by a decrease of Ecto-5'-Nucleotidase endocytosis, without modification of Ecto-5'-Nucleotidase association with detergent-resistant membranes. Finally, whereas cholesterol content was unmodified, hypoxia induced a time-dependent increase of saturated fatty acids in SVARECs, which was reversed by reoxygenation, in parallel to Ecto-5'-Nucleotidase stimulation. Incubation of normoxic cells with palmitic acid enhanced Ecto-5'-Nucleotidase activity and cell surface expression. In conclusion, hypoxia enhances cell surface expression of Ecto-5'-Nucleotidase in endothelial cells. This effect could be supported by a decrease of Ecto-5'-Nucleotidase endocytosis through modification of plasma membrane fatty acid composition.     

The role of transforming growth factor beta-2, beta-3 in mediating apoptosis in the murine intestinal mucosa

BACKGROUND & AIMS: Apoptosis is especially relevant in the gastrointestinal tract because the mammalian intestinal mucosa undergoes continual epithelial regeneration. Most recently, we confirmed the proapoptotic role of endogenous transforming growth factor (TGF)-beta in the developing chick retina as well as in chick ciliary, dorsal root, and spinal motor neurons. In the present study, we determined to establish the role of TGF-beta2 and TGF-beta3 in mediating apoptosis in non-neuronal tissue by analyzing the intestinal mucosa of Tgfbeta2(+/-) and Tgfbeta3(+/-) heterozygous mice. METHODS: Intestinal localization of TGF-beta2 and TGF-beta3 isoforms and antiapoptotic molecules Bcl-xL and Bcl-2 was examined immunocytochemically and by Western blot analysis. Apoptosis was detected by enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and proliferation was detected by proliferating cell nuclear antigen stains. RESULTS: TGF-beta2 was detected in endocrine cells, whereas TGF-beta3 was predominantly found in goblet cells. Programmed cell death was significantly reduced in the intestinal mucosa of Tgfbeta2(+/-) and Tgfbeta3(+/-) heterozygous mice. This decrease in apoptosis was accompanied by an increase in villus length; proliferation, however, seemed to remain unchanged. The level of Bcl-xL and Bcl-2 was significantly up-regulated in Tgfbeta2(+/-) and Tgfbeta3(+/-) mice. CONCLUSIONS: Our data show that TGF-beta2 and TGF-beta3 play an important role in mediating apoptosis in the intestinal mucosa and regulating apoptosis-associated proteins Bcl-xL and Bcl-2 in vivo.     

Mapping genetic determinants of coronary microvascular remodeling in the spontaneously hypertensive rat

This study aimed to analyze the role of endothelial progenitor cell (EPC)-derived angiogenic factors and chemokines in the multistep process driving angiogenesis with a focus on the recently discovered macrophage migration inhibitory factor (MIF)/chemokine receptor axis. Primary murine and murine embryonic EPCs (eEPCs) were analyzed for the expression of angiogenic/chemokines and components of the MIF/CXC chemokine receptor axis, focusing on the influence of hypoxic versus normoxic stimulation. Hypoxia induced an upregulation of CXCR2 and CXCR4 but not CD74 on EPCs and triggered the secretion of CXCL12, CXCL1, MIF, and vascular endothelial growth factor (VEGF). These factors stimulated the transmigration activity and adhesive capacity of EPCs, with MIF and VEGF exhibiting the strongest effects under hypoxia. MIF-, VEGF-, CXCL12-, and CXCL1-stimulated EPCs enhanced tube formation, with MIF and VEGF exhibiting again the strongest effect following hypoxia. Tube formation following in vivo implantation utilizing angiogenic factor-loaded Matrigel plugs was only promoted by VEGF. Coloading of plugs with eEPCs led to enhanced tube formation only by CXCL12, whereas MIF was the only factor which induced differentiation towards an endothelial and smooth muscle cell (SMC) phenotype, indicating an angiogenic and differentiation capacity in vivo. Surprisingly, CXCL12, a chemoattractant for smooth muscle progenitor cells, inhibited SMC differentiation. We have identified a role for EPC-derived proangiogenic MIF, VEGF and MIF receptors in EPC recruitment following hypoxia, EPC differentiation and subsequent tube and vessel formation, whereas CXCL12, a mediator of early EPC recruitment, does not contribute to the remodeling process. By discerning the contributions of key angiogenic chemokines and EPCs, these findings offer valuable mechanistic insight into mouse models of angiogenesis and help to define the intricate interplay between EPC-derived angiogenic cargo factors, EPCs, and the angiogenic target tissue.     

Hypoxia paradoxically inhibits the angiogenic response of isolated vessel explants while inducing overexpression of vascular endothelial growth factor

This study was designed to investigate how changes in O₂ levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1–4 % O₂. Additionally, aortic neovessels preformed in atmospheric O₂ lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O₂. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signaling molecules including VEGFR2, NRP1 and Prkc-beta while upregulating expression of VEGFR1. Medium conditioned by hypoxic cultures exhibited angiostatic and anti-VEGF activities likely mediated by sVEGFr1. Hypoxia reduced expression of VEGFR1 and VEGFR2 in endothelial cells while upregulating VEGFR1 in macrophages and VEGF in both macrophages and mural cells. Thus, changes in O₂ levels profoundly affect the endothelial response to angiogenic stimuli. These results suggest that hypoxia-induced angiogenesis is fine-tuned by complex regulatory mechanisms involving not only production of angiogenic factors including VEGF but also differential regulation of VEGFR expression in different cell types and production of inhibitors of VEGF function such as sVEGFR1.     

Matrix metalloprotein-9 activation under cell-to-cell interaction between endothelial cells and monocytes: possible role of hypoxia and tumor necrosis factor-α

Matrix metalloproteinase (MMP)-9 plays an important role in cardiovascular events. However, the mechanisms underlying in vivo activation of MMP-9 are largely unknown. We investigated the secretion and activation of MMP-9 under a cell-to-cell interaction, and the effects of hypoxia and cytokine. Human umbilical vein endothelial cell (HUVEC) and THP-1 (human monocyte cell line) were cultured individually, or cocultured under normoxic and hypoxic conditions. In a coculture of HUVEC and THP-1, proMMP-9 secretion was increased twofold compared with individual culture of HUVEC and THP-1, whereas MMP-2 secretion was unchanged. The increase in proMMP-9 secretion was suppressed by antiadhesion molecule antibodies and mitogen-activated protein kinase inhibitors, PD98059 (MAPK/ERK kinase1 inhibitor) and SP600125 (Jun N-terminal kinase inhibitor). ProMMP-9 secretion was increased by tumor necrosis factor (TNF)-α at 50 ng/ml (P < 0.05) but was not activated under normoxic (20%) conditions. ProMMP-9 in coculture was activated under hypoxic (<1%) conditions, and was potentiated by TNF-α (both P < 0.05). To further investigate the mechanism of hypoxia-induced MMP-9 activation, heat shock protein (Hsp)90, which was suggested to be related to MMP-9 activation, was measured by Western blot analysis. The ratio of Hsp90 to glyceraldehyde-3-phosphate dehydrogenase was increased in hypoxic (<1%) coculture conditions with TNF-α (P < 0.05). Treatment with geldanamycin and 17-DMAG (Hsp90 inhibitor) suppressed the active form of MMP-9. Cell-to-cell interaction between endothelial cells and monocytes promotes proMMP-9 synthesis and secretion. Hypoxia and inflammation are suggested to play an important role in activating proMMP-9, presumably via Hsp90.