ABSTRACT
Primary cell culture, techniques of gene transfection, gelatin zymography, and Western blot were used to investigate the effect of hypoxia on the secretion of MMP-2 and MMP-9 in pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), and the role of HIF-1. Our results showed that (1) after exposure to hypoxia for 24 h, the protein content and activity of MMP-2 in the PAEC medium as well as these of MMP-2 and MMP-9 in PASMC medium (P<0.01) decreased significantly in contrast to those in normoxic group (P<0.05); (2) after transfection of wild type EPO3'-enhancer, a HIF-1 decoy, the content and activity of MMP-2 and MMP-9 in hypoxic mediums became higher than those in normoxic group (P<0.01), while transfection of mutant EPO3'-enhancer didn't affect the hypoxia-induced down-regulation. It is concluded that hypoxia could inhibit the secretion and activity of MMP-2 and MMP-9 in PAEC and PASMC, which could be mitigated by the transfection of EPO3'-enhancer and that HIF-1 pathway might contribute to hypoxia-induced down-regulation of MMP-2 and MMP-9.
ABSTRACT
Wild type fragment of erythropoietin(Epo)3'-enhancer (W18) and its mutant type fragment (M18) were synthesized. Primary cultures of endothelial cells of different sources (ECs) and human umbilical venous endothelial cell line (HUVEC) as well as the primary culture of pulmonary artery smooth muscle cells (PASMC) were transfected with either W18 or M18. RT-PCR was performed to detect mRNA and investigate the effect of Epo3'-enhancer fragment on the hypoxia- induced gene expression in ECs. Electrophoretic mobility shift assay(EMSA) was used to test DNA-binding activity of extracted nuclear protein. ［3H］-TdR incorporation, MTT test and flow-cytometry were used to determine the proliferation of PASMC and the effect of Epo3'-enhancer fragment on it. The results showed as follows:(1)The OD value of COX-2 mRNA expressed in hypoxic rat aortic EC was 2.34±0.32, the OD values of COX-2 and VEGF mRNAs expressed in hypoxic EC of pulmonary microvasculature were 1.78±0.21 and 4.71±0.52, the OD value of TXS and ET-1 mRNAs in hypoxic HUVEC were 13.01±4.27 and 1.01±0.05, they were all higher than their counterpart normoxic group (P＜0.05). If the cells were pretransfected with W18, the OD values in these hypoxic groups became 1.28±0.25,0.77±0.09, 2.29±0.41, 4.88±1.05. and 0.51±0.15, respectively, just as low as those of the relevant normoxic groups; (2)The conditioned medium of hypoxic pulmonary artery endothelial cell (PAEC) caused proliferation of PASMC, the cpm values of ［3H］-TdR incorporation was 917.00±527.11, higher than that of normoxic control (P＜0.05). In addition, it was found by using flow-cytometry an increase in the percentage of cells of phases S and G2/M in PASMC incubated in hypoxic EC conditioned medium in comparison with normoxia group and group pretransfected with W18; (3)Hypoxia could induce proliferation of PASMC directly, transfection of W18 could decrease its effect. Their cpm values were 829.50±228.10 and 497.00±52.45, respectively(P＜0.05).The results of MTT test was similar; (4)The inducer of HIF-1, CoCl2 could increase the expression of COX-2 and TXS mRNA in HUVEC, which could also be inhibited by W18 but not by M18;(5)The HIF-1 DNA binding activity was found in hypoxic HUVEC in EMSA with 32 P labeled W18, but not found with 32 P M18. These results suggest that there might be a common pathway in hypoxic responses of different cells, i.e. regulating the transcription of genes by binding of HIF-1 to a sequence similar to Epo3'-enhancer.
ABSTRACT
AIM: To detect the role of cyclic nucleotides in the alleviation of hypoxic pulmonary vasoconstriction (HPV) in chronic hypoxic animals. METHODS: The intracellular cAMP and cGMP of the cultured porcine pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were assayed by RIA. The length of single PASMC during acute hypoxia was measured by imaging analysis system. RESULTS: The basal levels of cAMP and cGMP in PASMC and cGMP in PAEC of Chronic hypoxic groups decreased remarkably compared with normoxic groups ( P
ABSTRACT
Alteration of intracellular free calcium during hypoxia was studied in cul-tured porcine pulmonary arterial endothelial cells. Intracellular free calcium was measuredwith the fluorescent prob, Fura-2/AM. It was found that hypoxia induced by bubbling N_2 to cells suspension increased intracellular free calcium of endothelial cells by 81?21%(n=8, P
ABSTRACT
Roles of sympathicus, sensory neuropeptides (SNP), metabolites of cyclooxygenase, metabolites of lipoxygenase, endothelium derived relaxing factor (EDRF), reactive oxygen (ROS) and potassium channels (PC) in the hypoxic pulmonary vasoconstriction (HPV) and hypoxic cerebral vasodilation (HCVD) were studied in intact rats, rabbits and dogs. Results were as follows: during hypoxia, the excitation of sympathicus results in a constriction of both pulmonary and cerebral vessels; SNP, EDRF and the opening of 4-AP sensitive PC caused the dilation of both of them; metabolites of lipoxygenase mediated HPV and HCVD, whereas metabolites of cyclooxygenase were their modulators; hypoxia induced blockade of the ATP sensitive PC mediated HPV, but had no effect on HCVD; reduction of O_2~+ in the lung might potentiate HPV, but had no effect on HCVD. It is suggested that the alteration of lipoxygenase metabolites, ROS and ATP sensitive PC are factors accounting for the difference in response of pulmonary and cerebral vassels to hypoxia.
ABSTRACT
Investigation of the roles of prostaglandins(PGs) and leukotrienes(LTs) inthe changes of hemodynamics and hypoxic pulmonary vasoconstriction(HPV) induced bychronic cigarette smoking in Wistar rats.After exposure to either smoke or room air, rats were anesthetized, the changes ofhemodynamics and hypoxic pulmonary vasoconstriction were measured. Blood samplesdrawn through the carotid arterial cannula were analyzed with radioimmunoassay(RIA)for PGs and bioassay for LTs in plasma. The results showed that cigarette smoking for onemonth did not change the pulmonary arterial pressure nor the pulmonary vascularresistance(PVR) in rats, while the pressure response of pulmonary vessels to hypoxiawas reduced significantly, which could be abolished by indomethacin. Cigarette smokingcould increase the concentration of 6-Keto-PGF_1?but not that of TXB_2, in arterial plasma,during hypoxia and it reduced the plasma level of LTs as well. These results suggestedthat chronic cigarette smoking might have reduced HPV by two ways: i. e. increasing theptoduction of vasodilative PGs, especially PGI_2, and reducing LTs production duringhypoxia.
ABSTRACT
The alteration of hypoxic pulmonary vasoconstriction (HPV) induced bycigarette smoking was studied in Wistar rats, piglets and in humans. The percentage changeof pulmonary vascular resistance (△PVR%) and the amplitude of the systolic wave inimpedance pneumorheogram (△H%) were used to estimate the strength of HPV. It was observed that immediately after acute cigarette smoking, HPV in rats was in-creased (△PVR% from 55. 0?15. 6% to 102. 3?12. 4%), which was mainly mediated byleukotrienes (LTs); whereas HPV in piglets was decreased (△PVR% from 65.2?12. 5%to 55.9?9. 8%), which was mainly mediated by ? adrenergic receptors, and HPV inhumans was also increased (△H% from 20. 6? 2. 6% to 31. 1?4. 1%), in which prostag.landins and leukotrienes may play the role of mediators. However, after one-month ciga- rette smoking, the HPV of rats was significantly lowered (△PVR% 11. 4?1. 6%). Anincrease in vasodilative prostaglandins synthesis and a decrease in leukotrienes synthesismight haye contributed to this alteration of HPV.
ABSTRACT
Acute hypoxic pulmonary vasoconstriction (HPV) plays an important role in physiological regulation. The purpose of this study was to explore the effects of Ilexonin A on hemodynamics and HPV, and their relation to prostaglandins (PG_(?)) and leukotrienes (LT_(?)).The results show that the high pulmonary vascular resistance induced by Ilexonin A in normoxic rats may be partially caused by the increased LTs and have no relation to PGs. Ilexonin A may potentiate HPV at least by two ways: replacing the production of PGs (especially PGI_2) in hypoxic Wistar rats, and increasing LTs production.
ABSTRACT
TXB_2 and 6-keto-PGF_(1?) levels of arterial and venous plasma in Wistar and4 Hilltep rats were measured to investigate the roles of TXA_2 and PGI_2 in the hypoxic pulmonary vasoconstriction(HPV) and responsiveness difference of pulmonary vessels to hypoxia between different strains of rats. The results showed that PGI_2 might play an important role in maintaining the low resistance of pulmonary circulation in these two strains of rats. TXA, increased during hypoxia might partially mediate HPV in Hilltop rats, on the contrary, PGI_2 augmented during hypoxia might modulate HPV in Wistar rats. This might be the major methanism responsible for the more intensive responsiveness of pulmonary vessels to hypoxia in Hilltop than in Wistar rats.