Hypoxia Down-regulates Secretion of MMP-2, MMP-9 in Porcine Pulmonary Artery Endothelial and Smooth Muscle Cells and the Role of HIF-1 / 华中科技大学学报（医学）（英德文版）

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.

Subacute Mild Hypoxia Increases Histamine-stimulated Calcium Oscillation Frequency in Pulmonary Artery Endothelial Cells / 生物化学与生物物理进展

Calcium oscillation may regulate gene transcription in a frequency-decoding manner during agonist stimulation,which provides an indicator of transcription level in cells. To determine whether persistent exposure to hypoxia may sensitize or blunt cell response to histamine, the effects of 24 h subacute mild hypoxia on histamine-stimulated calcium oscillation frequency were examined in pulmonary artery endothelial cells (PAECs). The results are: (1) 24 h subacute mild hypoxia significantly increased the histamine-stimulated calcium oscillation frequency in PAECs. The averaged frequency of calcium oscillation in posthypoxic PAECs was significantly higher than that in normoxic ones. (2) NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI, 10 μmol/L), abolished histamine-stimulated calcium oscillations both in normoxic and posthypoxic PAECs. (3) Xanthine oxidase inhibitor, oxypurinol (100 μmol/L), did not affect the calcium oscillation kequency in normoxic PAECs. However, it significantly decreased the elevation of calcium oscillation frequency in posthypoxic PAECs. These results demonstrated that, during pulmonary disease related to persistent hypoxia,PAECs become more sensitive to histamine. During histamine stimulation, NADPH oxidase plays a critical role in generating calcium oscillations, while xanthine oxidase may contribute to, at least in part, the increase of calcium oscillation frequency in posthypoxic PAECs.

Inhibition of expression of hypoxia-inducible factor-1alpha mRNA by nitric oxide in hypoxic pulmonary hypertension rats / 华中科技大学学报（医学）（英德文版）

In order to study the effect of nitric oxide (NO) on the expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) mRNA in hypoxic pulmonary hypertension (HPH) rats, 30 healthy male Wistar rats were randomly divided into normoxic control group, chronic hypoxic group and hypoxia plus L-arginine (L-Arg) group. The animal model of HPH was developed. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The HIF-1alpha mRNA expression levels were detected by in situ hybridization (ISH) and semiquantitative RT-PCR. It was found that after 14 days hypoxia, the mPAP in normoxic control group (17.6 +/- 2.7 mmHg, 1 mmHg=0.133 kPa) was significantly lower than that in chronic hypoxic group (35.8 +/- 6.1 mmHg, t=0.2918, P<0.05) and mPAP in chronic hypoxic group was higher than that in hypoxia plus L-arginine group (24.4 +/- 3.8 mmHg, t=0.2563, P<0.05). ISH showed that the expression of HIF-1alpha mRNA in the intraacinar pulmonary arteriolae (IAPA) in normoxic control group (0.1076 +/- 0.0205) was markedly weaker than that in chronic hypoxic group (0.3317 +/- 0.0683, t=3.125, P<0.05) and that in chronic hypoxic group was stronger than that in hypoxia plus L-arginine group (0.1928 +/- 0.0381, t=2.844, P<0.05). RT-PCR showed that the content of HIF-1alpha mRNA in chronic hypoxic group (2.5395 +/- 0.6449) was 2.16 times and 1.75 times higher than that in normoxic control group (1.1781 +/- 0.3628) and hypoxia plus L-arginine group (1.4511 +/- 0.3981), respectively. It is concluded that NO can reduce the mPAP by the inhibition of the expression of HIF-1alpha mRNA, which may be one of the mechanisms through which NO affects the pathogenesis of HPH.

Effects of 3,4-dihydroxyacetophenone on cytosolic calcium in pulmonary artery endothelial and smooth muscle cells during acute hypoxia / 华中科技大学学报（医学）（英德文版）

The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3,4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3,4-DHAP could attenuate the hypoxic elevation of [Ca2+]i only in PASMCs but not in PAECs. It is concluded that 3,4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.

Inhibition of expression of hypoxia-inducible factor-1alpha mRNA by nitric oxide in hypoxic pulmonary hypertension rats / 华中科技大学学报（医学）（英德文版）

In order to study the effect of nitric oxide (NO) on the expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) mRNA in hypoxic pulmonary hypertension (HPH) rats, 30 healthy male Wistar rats were randomly divided into normoxic control group, chronic hypoxic group and hypoxia plus L-arginine (L-Arg) group. The animal model of HPH was developed. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The HIF-1alpha mRNA expression levels were detected by in situ hybridization (ISH) and semiquantitative RT-PCR. It was found that after 14 days hypoxia, the mPAP in normoxic control group (17.6 +/- 2.7 mmHg, 1 mmHg=0.133 kPa) was significantly lower than that in chronic hypoxic group (35.8 +/- 6.1 mmHg, t=0.2918, P<0.05) and mPAP in chronic hypoxic group was higher than that in hypoxia plus L-arginine group (24.4 +/- 3.8 mmHg, t=0.2563, P<0.05). ISH showed that the expression of HIF-1alpha mRNA in the intraacinar pulmonary arteriolae (IAPA) in normoxic control group (0.1076 +/- 0.0205) was markedly weaker than that in chronic hypoxic group (0.3317 +/- 0.0683, t=3.125, P<0.05) and that in chronic hypoxic group was stronger than that in hypoxia plus L-arginine group (0.1928 +/- 0.0381, t=2.844, P<0.05). RT-PCR showed that the content of HIF-1alpha mRNA in chronic hypoxic group (2.5395 +/- 0.6449) was 2.16 times and 1.75 times higher than that in normoxic control group (1.1781 +/- 0.3628) and hypoxia plus L-arginine group (1.4511 +/- 0.3981), respectively. It is concluded that NO can reduce the mPAP by the inhibition of the expression of HIF-1alpha mRNA, which may be one of the mechanisms through which NO affects the pathogenesis of HPH.

Effects of 3,4-dihydroxyacetophenone on cytosolic calcium in pulmonary artery endothelial and smooth muscle cells during acute hypoxia / 华中科技大学学报（医学）（英德文版）

The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3,4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3,4-DHAP could attenuate the hypoxic elevation of [Ca2+]i only in PASMCs but not in PAECs. It is concluded that 3,4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.

Expression of hypoxia-inducible factor-1 alpha and endothelin-1 gene in hypoxic pulmonary hypertension / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) and endothelin-1 (ET-1) gene in hypoxic pulmonary hypertension (HPH).</p><p><b>METHODS</b>The animal model of HPH was replicated. The elastic fiber staining was applied to show the intraacinar pulmonary artery (IAPA). Radioimmunoassay (RIA) and in situ hybridization (ISH) were used for detection of HIF-1a and. ET-1.</p><p><b>RESULTS</b>ISH showed that HIF-1alpha mRNA was expressed in the IAPA of all hypoxic rat. The expression was stronger in the H14 d (0.256 9 +/- 0.046 8) and H28 d (0.225 8 +/- 0.045 3) groups than in the H5 d (0.1455 +/- 0.072 2) and control (0.110 9 +/- 0.022 4) groups (P < 0.05), the expression of ET-1 mRNA in the H14 d (0.412 2 +/- 0.078 3) and H28 d (0.368 4 +/- 0.072 9) groups was also stronger than that in the H5 d (0.201 7 +/- 0.034 9) and control (0.185 5 +/- 0.036 1) groups (P < 0.05). The amount of ET-1 in pulmonary arteial blood in the H14 d [(158.78 +/- 25.14) pg/ml] and H28 d [(142.93 +/- 23.38) pg/ml] groups was significantly higher than that in the H5 d [(79.68 +/- 12.54) pg/ml] and control [(65.37 +/- 10.82) pg/ml] groups (P < 0.05). The mean pulmonary arterial pressure (mPAP) in the H14 d [(34.0 +/- 5.8) mm Hg] and H 28 d [(29.0 +/- 4.7) mm Hg] groups was markedly higher than that in the H5 d [(19.0 +/- 3.5) mm Hg] and control [(17.0 +/- 2.8) mm Hg] groups (P < 0.05). A positive rank correlation existed between the mPAP and the amount of ET-1 (rs = 0.747, P < 0.05).</p><p><b>CONCLUSIONS</b>Expression of HIF-1alpha and ET-1 mRNA in IAPA increase under long-term hypoxic condition and both show consistent expression, indicating that the expression of HIF-1a and ET-1 gene contribute to pathogenesis of HPH.</p>

Effect of recombined sICAM-1 on the adhesion of leukocyte to pulmonary microvascular endothelial cell stimulated by PMA / 中国病理生理杂志

AIM:To explore the effect of recombined sICAM-1 on the adhesion of leukocyte to pulmonary microvascular endothelial cell. METHODS:Primary cultured rat pulmonary microvascular endothelial cells were used in this experiment. Leukocyte was labeled with 99　Tm-HMPAO.The effects of different dose of sICAM-1, CA7 and dimeric form of sICAM-1 (sICAM-1*CA7)were tested separately in cell adhesion .RESULTS:sICAM-1 and CA7 could not inhibit cell adhesion even at the concentration of 100 mg/L,while sICAM-1*CA7 at concentrations of 20 mg/L and 40 mg/L could inhibit 42% and 50% of cell adhesion respectively (P＜0.05). CONCLUSION:sICAM-1 has poor inhibitory effect on cell adhesion, probably due to monomeric form.

Influences of chronic hypoxia on the gene expression of Kv1.3,Kv2.1 and Kv3.1 induced by acute hypoxia in PASMC of rats / 中国病理生理杂志

AIM and METHODS: Total RNA was extracted from 6th rat subcultured pulmonary artery smooth muscle cells(PASMC) exposed to continual chronic hypoxia or normoxia and the effects of chronic hypoxia on the changes of Kv1.3,Kv2.1,Kv3.1 mRNA in cultured PASMC induced by acute hypoxia were studied by semiquantitative RT-PCR in vitro. RESULTS:①Kv1.3,Kv2.1,Kv3.1 genes were found to be expressed in PASMC of rats exposed either to hypoxia or normxia.②The expression of Kv2.1 and Kv3.1 in 6th subcultured of PASMC in normaxia group could be upregulated by exposure to acute hypoxia,the levels of Kv2.1 and Kv3.1 mRNA were significantly increased from 0.646?0.092, 0.782?0.104 to 1.059?0.134, 0.985?0.116,respectively (P

Effect of chronic hypoxia on [Ca~(2+)]_ i in pulmonary artery endothelial cells and smooth muscle cells under acute hypoxia / 中国病理生理杂志

AIM AND METHODS: Using Ca 2+ -sensitive fluorescent probe Fura-2,we measured the changes of _i in cultured rat pulmonary artery endothelial cells (PAEC) and porcine pulmonary artery smooth muscle cells (PASMC) from normoxic (NC group) or chronic hypoxic group (CH group) when they were exposed to acute hypoxia. RESULTS: The increase in _i in 6th passage of PASMC caused by acute hypoxia in CH group was significantly lower than that in the same passage of NC group (P

Changes of nuclear factor-?B and inducible nitric oxide synthase in hypoxic pulmonary hypertension / 中国病理生理杂志

AIM:To observe the changes of nuclear factor-?B (NF-?B) activity and inducible nitric oxide synthase (iNOS) expression in hypoxic pulmonary hypertension(HPH). METHODS:The rat model of HPH was used. The NF-?B activity and iNOS expression in lung tissue were determined by immunohistochemistry(IHC), in situ hybridization (ISH), RT-PCR and Western blot.RESULTS:ISH showed that iNOS mRNA expression in intraacinar pulmonary arteriole (IAPA) in H28 d group(hypoxic treatment for 28 days) was stronger than that in normal group, H5d group and H14 d group. RT-PCR showed that the iNOS mRNA in H28 group was 2.1 times, 1.9 times and 1.8 times higher than that in normal group, H5d group and H14 d group, respectively. The nucleic anti-NF-?B stain was observed in H28 d group, which was significantly stronger, but the I-?B amount was 2.8 times, 2.7 times and 2.5 times lower than that in normal group, H5d group and H14 d group, respectively. CONCLUSION: The activity of NF-?B was correlated with the hypoxic pulmonary vessel structural remodeling and iNOS expression.

Role of K~+ channels in attenuation of pulmonary vasoreactivity following chronic hypobaric hypoxia in rats / 中国病理生理杂志

AIM and METHODS: To determine the role of different K +-channels in attenuation of vasoreactivity of intrapulminary artery rings induced by chronic hypoxia. RESULTS: ①Acute hypoxia-induced pulmonary vasoconstriction(HPV) could be significantly attenuated by chronic hypobaric hypoxia for 15 days and for 30 days. ②HPV could be significantly potentiated by ATP-sensitive K +-channel (K ATP ) blocker or Ca 2+ -activated K +-channel (K Ca )blocker, and the potentiated scope in chronic hypoxic group was much higher than that observed in control group. ③ Delayed rectifier K +-channel (K DR )blocker showed no effect on HPV in both control group and chronic hypoxic group.CONCLUSIONS: Both K ATP and K Ca play an important modulating role in HPV and it's potentiation may be a critical mechanism for the attenuated vasoreactivity to acute hypoxia; following chronic hypobaric hypoxia.

Role of cyclic nucleotides in the acute hypoxic responses of hypoxic subcultured porcine pulmonary arterial smooth muscle and endothelial cells / 中国病理生理杂志

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

Effects of hydrogen peroxide on cyclooxygenase-2 expression in pulmonary arterial endothelial cells and the role of CaMKⅡ / 中国病理生理杂志

AIM: To investigate if hydrogen peroxide may alter COX-2 gene expression in pulmonary artery endothelial cells(PAECs) and how CaMKⅡ functions in this process.METHODS: Cultured pulmonary arterial endothelial cells were treated with different concentrations of hydrogen peroxide for different durations.The cells survival rates were measured by CCK-8 after the cells were treated by hydrogen peroxide.The level of COX-2 mRNA and protein were measured by RT-PCR and Western blotting,respectively.RESULTS: The results showed that hydrogen peroxide up-regulated COX-2 mRNA and protein levels in a concentration-and time-dependent manners.Incubation with 100 ?mol/L H2O2 for 4 h increased COX-2 mRNA and protein level to 256.01%?22.36%(P

Role of three kinds of potassium channel in airway hyperresponsiveness of asthmatic guinea pigs / 中国病理生理杂志

AIM: To investigate the role of Ca2+ - activated, delayed - rectifier and ATP sensitive K+ channel (KCa, Kdr, KATP) in airway hyperresponsiveness of asthmatic guinea pigs. METHODS: The method of recording the tone of isolated trachea rings was performed, and the changes of dose-response curves of trachea rings to histamine caused by different K+ channel blockade were investigated. RESULTS: (1) After inhibition of KCa, by tetraethylammonium (TEA) , the dose - response curve of trachea rings to histamine did not change in control group, while the maximal contraction of trachea rings to 10-4 mol/L and 10-3 mol/L histamine decreased significantly ( P

Cellular oxygen sensors and signal transduction / 中国病理生理杂志

Hypoxia is a commenest pathological process. The cellular oxygen sensors and signal transduction involved in hypoxic responses are so far not fully elucidated. There are many theories. Perhaps the oxygen sensors are different among different kinds of cells, and between acute and chronic hypoxic responses. The mitochondria cytochrom-oxidase-H_2O_2 might be the principal pathway leading to the constrictive response of pulmonary smooth muscle cells to hypoxia. The heme protein - reactive oxygen pathway might mediate the response of glomus cells in carotid body to hypoxia. The NADPH oxidase might be the oxygen sensor in airway chemoreceptor. As for chronic hypoxic responses, the oxygen dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation has been paid great attention in recent years.

Effects of exercise stress on cigarette smoking-induced downregulation of BK_(Ca) and Kv1.5 expression in rat bronchial smooth muscle cells / 中国病理生理杂志

AIM: To investigate the effect of exercise stress on chronic cigarette smoking-induced downregulation of expression of large-conductance calcium-activated potassium channel (BK_(Ca)) and voltage-dependent delayed rectifier potassium channel (Kv1.5) in rat bronchial smooth muscle cells. METHODS: Rats were divided into 3 groups: normal control, smoking control and smoking plus exercise training group. The alteration of airway responsiveness and plasma cortisol level were detected, and potassium channel expression and pathological changes in lung tissue were determined with HE staining, immunohistochemistry, in situ hybridization and Western blot techniques. RESULTS: (1) Cigarette smoking induced an increase in airway responsiveness, smoking plus exercise lead to a decrease in airway responsiveness in contrast to smoking control group; (2) Plasma level of cortisol determined immediately after exercise was higher than that determined before exercise; (3) HE staining showed that there was severe chronic pulmonary inflammatory response in smoking control group, which was slight in the smoking plus exercise group; (4) The protein and mRNA expression of BK_(Ca) in cigarette smoking group were less than that in control group in BSMC, the mRNA expression of BK_(Ca) in exercise group were higher than that in smoking group; (5) The protein and mRNA expression of Kv1.5 in smoking group were less than that in control group in BSMC, and expression of Kv1.5 in exercise group was higher than that in smoking group in bronchioli. CONCLUSION: Proper exercise training can increase the expression of potassium channel BK_(Ca) and Kv1.5, and increase the cortisol secretion, which may contribute to the decreasing of airway hyperresponsiveness induced by cigarette smoking. [

Chronic hypoxia altered the expression of ET-1 mRNA in response to acute hypoxia in pulmonary artery endothelial cells / 中国病理生理杂志

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: In situ hybridization 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.

Role of K~+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats / 中国病理生理杂志

AIM: To investigate the role of K + channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS: Blockers of three kinds of K + channels, 4-AP(voltage dependent K + channel blocker), TEA(Ca 2+ activated K + channel blocker)\, GLIB(ATP sensitive K + channel blocker) were used in isolated perfused rat lungs to detect the role of K + channels in HPV. RESULTS: In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION: The opening of Kv, K Ca , K ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.

Effect of recombined sICAM-1 on the adhesion of leukocyte to pulmonary microvascular endothelial cell stimulated by PMA / 中国病理生理杂志

AIM: To explore the effect of recombined sICAM-1 on the adhesion of leukocyte to pulmonary microvascular endothelial cell. METHODS: Primary cultured rat pulmonary microvascular endothelial cells were used in this experiment. Leukocyte was labeled with 99 Tm-HMPAO.The effects of different dose of sICAM-1, CA7 and dimeric form of sICAM-1 (sICAM-1?CA7)were tested separately in cell adhesion .RESULTS: sICAM-1 and CA7 could not inhibit cell adhesion even at the concentration of 100 mg/L,while sICAM-1?CA7 at concentrations of 20 mg/L and 40 mg/L could inhibit 42% and 50% of cell adhesion respectively ( P