Hypoxia induces myofibroblast formation and stimulates production of collagen I in myofibroblasts through ERK1/2 pathway

AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen I in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia (1% O2) or normoxia (21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1α (HIF-1α) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of α-smooth muscle actin (α-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1α in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase (ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points (2 h,4 h and 6 h).The distribution of HIF-1α in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1α protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1α was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of α-SMA protein increased in NRK-49F under hypoxia for 12 h (187%±32%,P<0.05).The level of collagen I protein in culture medium was increased in hypoxia treated myofibroblasts at 6 h (171%±27%,P<0.05) and 12 h (256%±61%,P<0.05).Collagen I mRNA expression was increased in cells under hypoxia condition for 4 h (189%±28%,P<0.05) and 6 h (221%±44%,P<0.05).The activities of MMP-2 and MMP-9 in the supernatant medium were not significantly changed at different experimental time points between the normoxic and hypoxic conditions.Activation of ERK1 /2 occurred as early as 15 min,sustained the high level at 30 min and 60 min and was back to the baseline level at 2 h.Blockade of ERK activation with PD98059 abolished hypoxia-induced expressions of collagen I protein.CONCLUSION: Hypoxia contributes to the renal interstitial fibrosis through inducing formation of myofibroblasts and stimulating the production of collagen I in myofibroblasts.     

Regulation of hypoxic response elements to expression of hVEGF165 gene transferred to cardiomyocytes under anoxic and normoxic conditions

AIM:To investigate effects of hypoxic response elements (HRE) on expression of hVEGF165 gene transferred to myocardiocyte under anoxic and normoxic conditions in vitro.METHODS:Myocardiocytes from neonatal SD rats were isolated and cultured in serum-free medium.The r-AAV vector packaged with 293T cells was used to transfer cultured myocardiocytes.Myocardiocytes were divided into eight groups: Group Ⅰ: myocardiocytes were cultured for 24 hours under normoxic conditions as control;Group Ⅱ: cells was cultured under hypoxia for 8 hours;Group Ⅲ: infected cells were cultured for 24 hours under normoxic conditions;Group Ⅳ: infected cells were cultured under hypoxia for 8 hours;GroupⅤ: infected cells were cultured under hypoxia for 8 hours and normoxia for 4 hours;GroupⅥ: infected cells were cultured under hypoxia for 8 hours and normoxia for 8 hours;GroupⅦ: infected cells were cultured under hypoxia for 8 hours and normoxia for 12 hours;Group Ⅷ: infected cells were cultured under hypoxia only for 20 hours.hVEGF165 protein was quantified from culture medium using ELISA,hVEGF165 protein in myocardiocytes was detected with immunofluorescence and expression of hVEGF165 mRNA was also determined by RT-PCR.RESULTS:95% myocardiocytes showed beating rhythmically with 86% of cTn-I positive staining.87% virual transferred efficiency was achieved.The contents of hVEGF165 protein in group Ⅳ,Ⅴ and Ⅷ were higher than those in other groups (P<0.01).Immunofluorescence positive cells were observed in group Ⅳ,Ⅴ and group Ⅷ.RT-PCR revealed that a 484 bp strip was also found in the same groups.CONCLUSION:rAAV-HRE9-hVEGF165 vector infected cultured myocardiocyte successfully.Under hypoxia,expression of hVEGF165mRNA and hVEGF165 protein were regulated by HRE,whereas under normoxic conditions the expression of hVEGF165 mRNA and hVEGF165 protein were ceased.     

Effects of hypoxia on the expression of heparanase and invasiveness of SKOV3 ovarian cancer cell line

AIM: To evaluate the effects of hypoxia on the expression of heparanase and the invasiveness of SKOV3 ovarial carcinoma cell line. METHODS: SKOV3 cells were incubated at either normoxia (37 ℃, 5％CO2, 21％O2) or hypoxia (37 ℃, 5％CO2, 1％O2) condition for 12 h, 24 h and 36 h. RT-PCR and Western blotting were used to detect mRNA and the protein expressions of heparanase under different conditions. Cell invasiveness was measured by matrigel invasion assay. RESULTS: Compared to normoxia group, the heparanase mRNA expression level in hypoxia group was increased and in 12 h hypoxia group was the highest. The heparanase protein expression in hypoxia group was also significantly increased (P<0.01) and the expression of heparanase in hypoxia group was also different (P<0.05). Compared to normoxia group, the level of cell invasion was markedly increased in 12 h, 24 h and 36 h groups (P<0.05). During 12-36 h hypoxia period, the increase in hypoxia-induced invasiveness in SKOV3 cell line showed a time-dependent manner (P<0.05). Meanwhile, there was a positive correlation between the expression of HPA and the invasiveness of SKOV3 cells (r=0.8530, P<0.05). CONCLUSION: Invasion of SKOV3 cells in hypoxia condition correlates with heparanase level. Hypoxia plays an important role in the augmentation of the heparanase expression and the invasiveness of human ovarian cancer.     

Effect of sulodexide on apoptosis of human dermal microvascular endothelial cells exposed to hypoxia

AIM: To investigate the effect of sulodexide (SDX) on the apoptosis of human dermal microvascular endothelial cells (HDMECs) exposed to hypoxia and its underlying mechanism. METHODS: The HDMECs were cultured and divided into normoxia control group cultured under normoxic condition; hypoxia control group cultured in a humid incubator maintained at 37℃ with 5% CO₂ and 1% O₂ for 24 h; treatment groups treated with SDX at 0.25, 0.5 and 1 LSU/mL for 24 h under hypoxic condition. The cell viability was measured by CCK-8 assay. The apoptotic rate of the HDMECs was analyzed by flow cytometry. The activity of caspase-3 in HDMECs was examined by caspase-3 activity assay kit. The expression of pro-apoptotic factor P53, caspase-3, Bax and anti-apoptotic factor Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: SDX increased the viability of HDMECs exposed to hypoxia, but also decreased the apoptosis. Furthermore, SDX down-regulated the expression of pro-apoptotic factor P53, Bax and caspase-3 at mRNA and protein levels as well as the activity of caspase-3, while the expression of anti-apoptotic factor Bcl-2 was up-regulated. CONCLUSION: SDX significantly increases the viability and decreases the apoptosis of HDMECs exposed to hypoxia. Inhibition of the mitochondrial apoptosis pathway may be involved in the underlying mechanism.     

Effects of tanshinone IIA on proliferation,apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia

AIM:To investigate the effects of tanshinone IIA (Tan IIA) on proliferation, apoptosis and its molecular mechanism in human hepatoma HepG2 cells under hypoxic condition. METHODS:Hypoxia model was established by treatment with cobalt chloride (CoCl2). The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups. After HepG2 cells were incubated with different concentrations of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell proliferation was determined by MTT assay. After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining. The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h. RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner. Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose- and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1α and VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incubated under hypoxia for 48 h. The protein expression of HIF-1α and VEGF were decreased with the increase in the concentration of Tan IIA under hypoxia. The protein expression of wild-type P53 was increased with the increase in the concentrations of Tan IIA under hypoxia. CONCLUSION: Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1α and VEGF and up-regulation of wild-type P53.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

Oxysterols downregulate tissue inhibitor of metalloproteinase -1 expression in vascular smooth muscle cells

AIM:To investigate the effects of oxysterols on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in vascular smooth muscle cells. METHODS:Rabbit aortic vascular smooth muscle cells were culturedin vitroand incubated with cholesterol, Triol and 25-hydroxycholesterol (25-OH), respectively. Slot blot was used to detect the mRNA expression level of TIMP-1 in vascular smooth muscle cells (VSMCs); meanwhile the protein expression level of MMP-9 and TIMP-1 was detected by immunohistology. RESULTS:Triol and 25-OH inhibited the expression of TIMP-1 compared with control and cholesterol, but have no effect on expression of MMP-9. CONCLUSION:Both Triol and 25-OH downregulated TIMP-1 expression in VSMCs.     

Expression of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and collagen type IV in lung of rats with multiple organ dysfunction syndrome

AIM: To determine the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of me-talloproteinase-1 (TIMP-1) and collagen type IV (IV-C) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. METHODS: Adult male Sprague-Dawley (SD) rats (n=40) were randomly divided into sham control group and cecal ligation and puncture (CLP) model group. The rats in CLP group were divided into 4 subgroups as different intervals (6 h, 12 h, 24 h and 48 h), and there were 8 rats in each group. The rat model of MODS was established by CLP. All rats were sacrificed at various intervals. The functions of the liver, kidney and lung were determined by blood biochemical and blood gas analysis. The morphological changes of the lung tissues were observed with HE staining. The serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, MMP-9 and TIMP-1 were measured by ELISA. The expression of MMP-9 and TIMP-1 in the lung tissues was detected by RT-PCR and immunohistochemistry, and the expression of IV-C in the lung tissues was detected by immunofluorescence and Western blot. RESULTS: Compared with sham control group, the functions of the liver, kidney and lung were damaged at different degrees in model groups. No histopathological change in the lung tissues of sham control group was found, and the lung injury was serious in model groups. Compared with sham control group, the serum levels of TNF-α, IL-1β, MMP-9 and TIMP-1 in model groups increased significantly (P<0.05) and peaked at the interval of 12~24 h after modeling (P<0.01). The expression of MMP-9 and TIMP-1 in the lung tissues of model groups increased, and peaked at 12 and 24 h, respectively (P<0.01). The protein level of IV-C in MODS 6 h group was not changed as compared with control group, while that at the interval of 12~48 h after modeling was significantly decreased and dropped to the lowest at 24 h (P<0.01). CONCLUSION: MMP-9 and TIMP-1 play important roles in lung injury of MODS rats by regulating the synthesis and decomposition of IV-C which is the main component of extracellular matrix.     

Expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in lung tissue of hypercapnia rat model

AIM: To study the expression of matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2) and the tissue inhibitor of metalloproteinase(TIMP-1) in the lung tissue of the hypercapnia rat.METHODS: Forty Wistar rats were randomly divided into a control group (group A, n=20) and hypercapnia group (group B, n=20). Group B received mix gas exposure (6% CO2, 21% O2, 72% N2) 7 h daily for 4 weeks. The parameters we would examine were as follow: arterial blood gas; the mean pulmonary artery pressure;MMP-2,MMP-9, TIMP-1, and NE activity in lung tissue. Masson pigmentation of elasticity fibre was analyzed by computer image analyzer. Histopathological changes of lung tissue were observed under light microscope. The protein expression of MMP (MMP-2, MMP-9) and TIMP (TIMP-1) in lung tissue were determined by immunocytochemistry.RESULTS: Decompensate respiratory acidosis (pH=7.20±0.04, PaCO2=7.84±0.15) developed in group B. The mean pulmonary artery pressure were similar between groups B and A (P>0.05). Tissue edema in the lung, endothelial cell damage of the small blood vessels, pulmonary micro thrombus formations and increased pulmonary capillary permeability were observed in group B. NE activity increased significantly (P<0.01). However, no significant change of MMP-2, MMP-9, TIMP-1 activity was found in group B and group A (P>0.05). There was significant decrease in the relative content of elasticity fibre in lung tissue in group B compared to group A (P<0.01). The expression of MMP-2 protein in the lung tissue of group B was lower than that in group A (P<0.01), but the expression of both MMP-9 and TIMP-1 proteins in the lung tissue in group B were higher than those in group A (P<0.01).CONCLUSION: Hypercapnia rat model is successfully reproduced by exposure of animals to the mix gas exposure (6% CO2, 21% O2, and 72% N2). The pulmonary artery pressure is not affected by hypercapnia. High concentration of CO2 causes increase of NE activity and decrease in the relative content of elasticity fibre. High concentration of CO2 causes the increase of MMP-2 protein expression and decrease in the MMP-9 and TIMP-1 protein expression.     

Response of mitochondrial reactive oxygen species in pulmonary arterial smooth muscle cells under acute hypoxic condition

AIM:To observe the response of mitochondrial reactive oxygen species (ROS) in rat pulmonary artery smooth muscle cells (PASMCs) under acute hypoxic condition. METHODS:The cultured PASMCs were under normoxic (35 ℃, 5% CO2, 21% O2, 74% N2) or acute hypoxic (35℃, 5% CO2, 1% O2, 94% N2) condition. The cells were incubated with molecular probes chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and RedoxSensor Red CC-1 to detect the ROS generation by laser scanning confocal microscopy. The mitochondria were isolated and mitochondrial inhibitors were used to detect the ROS generation functional unit sites by spectrophotometry under acute hypoxic condition. RESULTS:Under acute hypoxic condition, the intracellular ROS was significantly increased in hypoxia group with 3.35 folds higher of H2O2 than that in normoxia group. The contents of H2O2 and O-·2 in hypoxia group were 1.61 folds higher than those in normoxia group. Compare with hypoxia goup, pretreatment with the mitochondrial electron transport chain (ETC) complex I inhibitor MPP, the complex II inhibitors NPA and TTFA as well as the complex III pre-ubisemiquinone site inhibitor myxothiazol all remarkably reduced hypoxia-induced increase in ROS generation in PASMCs (reduced by 60%, 73%, 75% and 61%, respectively, P<0.01), whereas the complex III postubisemiquinone site inhibitor antimycin A and the complex IV inhibitor NaN3 had no effect on hypoxia-induced increase in ROS generation (increased by 13% and 9.1%, respectively, P>0.05). Direct detection of mitochondrial ROS showed the same results as the intracellular ROS. CONCLUSION: The intracellular ROS increases significantly in rat PASMCs under acute hypoxic condition. The mitochondrial ETC complex I, complex II and complex III pre-ubisemiquinone sites increase ROS generation, whereas the complex III postubisemiquinone site and complex IV do not produce this effect under acute hypoxic condition.     

PAI-1,TIMP-1 gene expression in renal tubulointerstitial fibrosis of ureteral obstruction and the interfering effects of HGF treatment

AIM: The aim of this study was to determine the relationship between PAI-1,TIMP-1 gene expression and renal tubulointerstitial fibrosis(TIF) of ureteral obstruction ,and the interfering effects of hepatocyte growth factor (HGF) treatment. METHODS: Sixty rats were divided into normal control, sham operation,unilateral ureteral obstruction(UUO),and rhHGF treated groups (received 0.5 mg·kg^-1·d^-1 HGF for twenty-one days), and were sacrificed at postoperative day 3, 7, 14, 21. The levels of PAI-1, TIMP-1 mRNA were measured by RT-PCR. MMP2 and MMP9 activities were detected by substrate zymography, and renal fibrosis was assessed by measuring tissue hydroxyproline. RESULTS: Compared to controls, expression levels of PAI-1,TIMP-1 mRNA were significantly increased in UUO rats, and this was accompanied by decreased activities of MMP2 and MMP9 and increase in tissue hydroxyproline content. HGF treatment significantly decreased expressions of PAI-1, TIMP-1 mRNA, increased MMP2 and MMP9 activities,and decreased tissue hydroxyproline content in the obstructive kidney. CONCLUSIONS: These results indicate that the increases in PAI-1, TIMP-1 mRNA expression may be the major cause of sustained decreased matrix degradation during the development of tubulointerstitial fibrosis after unilateral ureteral obstruction. rhHGF efficiently ameliorates renal tubulointerstitial injury by the reduction of PAI-1,TIMP-1 mRNA expression, and increasing MMP2, MMP9 activities.     

Proteomic study on mice hepatic cell membrane proteins after exposed to hypoxia

AIM: To identify the differential expression of mice hepatic cell membrane proteins after hypoxic exposure.METHODS: The hepatic cells of C57BL/6 mice were cultured for 8 h under hypoxic or normoxic conditions and the membrane proteins were extracted. The differentially expressed membrane proteins were separated by two-dimensional electrophoresis. The technique of matrix-assisted laser desorption/ionization mass time-of-flight spectrometry(MALDI-TOF-MS) was used to analyze the proteins.RESULTS: Compared with the proteins extracted from the cells under normoxic condition with a threshold of 1.5-fold, 28 differentially expressed proteins were identified in the proteins extracted from the cells under hypoxic condition according to the database NCBInr 20071130, in which 9 were down-regulated and 7 were up-regulated, including prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP.CONCLUSION: The results suggest that prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP might play important roles in sensing of cellular oxygen and the related signal transduction pathways.     

Inhibitory effect of notoginsenoside monomer R1 on activation of p38 MAPK signaling pathway induced by hypoxic hypercapnia in PASMCs

AIM: To explore the mechanism of notoginsenoside monomer R₁ (R₁) against hypoxic hypercapnia-induced pulmonary vasoconstriction (HHPV) by investigating the effect of R₁ on p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs) under the condition of hypoxia and hypercapnia. METHODS: Primary cultured PASMCs, which were isolated from Sprague-Dawley rats, were incubated in logarithmic growth phase from the 2nd to 5th generation with different concentrations (8, 40 and 100 mg/L) of R₁ under the condition of 6% CO₂ plus 1% O₂ for 24 h. The expression of p38 at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. RESULTS: The results of Western blotting and RT-PCR analysis indicated that the protein and mRNA expression levels of p-p38 MAPK were significantly higher in hypoxic hypercapnia group with DMSO control than those in normoxia control group (P<0.01). In R₁ treatment groups, the levels of p-p38 MAPK protein and p38 MAPK mRNA were markedly decreased (P<0.01) in a dose-dependent manner. CONCLUSION: p38 MAPK signaling pathway may mediate hypoxic hypercapnia pulmonary vasoconstriction in rats. Notoginsenoside monomer R₁ attenuates HHPV, which may be related to blockage of p38 MAPK signal pathway.     

Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

Effect of hypoxia on invasion and migration of lung carcinoma cells and its molecular basis

AIM: To investigate the effect of hypoxia on the invasion and migration of lung carcinoma cells. METHODS: Lung carcinoma cell H128 was exposed to normoxia (air, 5% CO₂), hypoxia (5% O₂,5% CO₂,90% N₂) or anoxia (95% N₂,5% CO₂) conditions for 48 hours. The migration ability of the cells was assayed by wound healing methods. The invasiveness ability was determined with HABM-HEM model. The cells exposed to hypoxia were inoculated under skin in nude mice, and then the growth of the tumor and the rate of metastasis to lymph node or lung were observed. The expression of E-cadherin and β1-integrin on the cells were also assayed by flow cytometry. RESULTS: Compared with normoxic group, the invasiveness and migration in hypoxic group were increased. The rates of tumorgenesis and metastasis to lung in anoxic group were evidently decreased. The expression of E-cadherin was decreased, and the expression of β1-integrin was increased in hypoxic group. In anoxia group, the invasiveness, migration, the expression of E-cadherin and β1-integrin were all decreased. CONCLUSIONS: Moderate hypoxia down-regulated the expression of E-cadherin, up-regulated the expression of β1-integrin, and increased the invasiveness and metastasis of carcinoma cells. Serious hypoxia decreased the expression of adhesive molecules, and the proliferation, invasiveness and migration were also decreased.     

Chronic hypoxia altered the expression of ET-1 mRNA in response to acute hypoxia in pulmonary artery endothelial cells

AIM:To investigate the expression of ET-1 mRNA in porcine pulmonary artery endothelial cells cultured in normoxic and chronic hypoxic conditions, and their different responses to acute hypoxia were also evaluated.METHODS:Insituhybridization and image -analysis system were used. RESULTS:Acute hypoxia enhanced the expression of ET-1 mRNA in both normoxic and chronic hypoxic group. The increment was more significant in the latter group.CONCLUSION:Chronic hypoxia increased the expression of ET-1 mRNA in response to acute hypoxia in porcine pulmonary artery endothelial cells.