Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.     

Effect of hypercapnia on lysyl oxidase-dependent collagen cross-linking and hypoxia-induced pulmonary hypertension in rat

AIM: To investigate the effect of hypercapnia on hypoxia-induced pulmonary hypertension and the changes of lysyl oxidase (LOX) and extracellular matrix collagen cross-links in the rat. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia group, hypoxia group, hypercapnia group and hypoxia+hypercapnia group. LOX activity was detected by fluorescence spectrophotometry. LOX protein expression was detected by immunohistochemistry and Western blot. The mRNA expression of LOX in the pulmonary artery was detected by real-time PCR. RESULTS: The levels of mean pulmonary artery pressure (mPAP), RV/(LV+S) and WA/TA in hypoxia group were significantly higher than those in normoxia group (P<0.01). Moreover, the levels of mPAP and RV/(LV+S) in hypoxia+hypercapnia group were significantly lower than those in hypoxia group (P<0.01). However, no significant difference of mPAP and RV/(LV+S) between hypercapnia group and normoxia group was observed. In hypoxia group, the collagen cross-links in the lung tissue was significantly higher than that in normoxia group and hypercapnia group (P<0.01). Importantly, collagen cross-links in the lung tissue of hypoxia+hypercapnia group was significantly lower than that in hypoxia group (P<0.01). There was no significant difference in collagen cross-links between hypercapnia group and normoxia group. The expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries of hypoxia group were significantly increased as compared with normoxia group (P<0.01). Furthermore, the expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries in hypoxia+hypercapnia group were lower than those in hypoxia group (P<0.01). CONCLUSION: Hypoxia not only up-regulates LOX but also promotes collagen cross-linking in the rat lung, which contributes to the development of pulmonary hypertension. Hypercapnia inhibits hypoxia-induced LOX expression and collagen cross-linking, therefore impairing the progress in hypoxia-induced pulmonary hypertension.     

Expression of ROCK I and TGF-β1 in pulmonary arterioles of rat with chronic thromboembolism pulmonary hypertension

AIM:To investigate the dynamic expression of Rho kinase (ROCK I) and transforming growth factor β₁ (TGF-β₁) in pulmonary arterioles of rat with chronic thromboembolic pulmonary hypertension. METHODS: Sixty-four male Wister rats were randomly divided into eight groups: beginning control group, embolism for 3 d, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks groups and end control group. The pulmonary thromboembolism (PTE) model was established by injecting thrombin into jugular vein two times in two weeks and each rat underwent peritoneal injection with tranexamic acid one time a day during experiment to prevent thrombolysis. The mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), relative medial thickness of small pulmonary arteries (PAMT) and vessel wall area/total area (WA/TA) were measured. The levels of ROCK I mRNA and TGF-β₁ protein in rat pulmonary artery were determined by in situ hybridization, immunohistochemistry and image analysis, respectively. RESULTS: mPAP, PAMT and WA/TA were higher respectively in embolism from 4 weeks group to 12 weeks group than those in beginning control group (mPAP: all P<0.01, PAMT and WA/TA: 4 weeks group P<0.05, 8 weeks group and 12 weeks group P<0.01). RVHI was elevated in 8 weeks group P<0.05, in 12 weeks group P<0.01. ROCK I mRNA and TGF-β₁ protein in pulmonary arterioles got the enhanced positive signals of in situ hybridization or immunohistochemistry staining with prolonging the time of rats with pulmonary thromboembolism. ROCKⅠ mRNA: embolism from 3 d group to 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01, TGF-β₁ protein: 1 week group and 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01. Linear correlation analysis showed that ROCK I mRNA and TGF-β₁ protein were positively correlated with mPAP, RVHI and vessel remodeling index (all P<0.01), ROCK I mRNA were positively correlated with TGF-β₁ protein (P<0.01). CONCLUSION:ROCK I and TGF-β₁ play a role in the pathogenesis of chronic thromboembolic pulmonary hypertension and pulmonary vessel remodeling. TGF-β₁ produces biological effect by active ROCK signal pathway.     

Effect of puerarin on monocrotaline-induced pulmonary hypertension and expression of chemokine CX3CL1/fractalkine

AIM: To observe the change of CX₃CL1/fractalkine (FKN) in the rats with monocrotaline-induced pulmonary hypertension, and to study the intervention of puerarin. METHODS: The pulmonary hypertension model was established in vivo by intraperitoneal injection of monocrotaline. Thirty male Sprague-Dawley rats (270-310 g) were randomly divided into 3 groups: control group (C), monocrotaline model group (M)and puerarin treatment group (M+P). The mean pulmonary arterial pressure (mPAP), mean right ventricular pressure (mRVP), mean carotid arterial pressure (mCAP) and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) were also detected. The structural changes of pulmonary arterioles were observed under optical microscope. Remodeling of lung blood vessels was determined by measuring the ratio of vessel wall area to total area (WA/TA) and the medium thickness of pulmonary artery (PAMT). The concentration of soluble fractalkine(sFKN) in plasma was measured by ELISA. The expression of FKN in the pulmonary artery wall was measured by immunohistochemistry. The mRNA level of FKN in the lung tissues was detected by RT-PCR.RESULTS: mPAP, mRVP, RV/(LV+S), WA/TA and PAMT in M group were higher than those in C group (P<0.01). RV/(LV+S), WA/TA and PAMT in M+P group were significantly lower than those in M group (P<0.01). No significant difference of mCAP among the 3 groups was observed. The levels of sFKN, FKN mRNA and FKN protein in M group were higher than those in C group (P<0.01), and the above data in M+P group were lower than those in M group(P<0.05). The serum level of sFKN had a positive correlation with PAMT and RV/(LV+S) (r＝0.719, r=0.685,respectively, P<0.01).CONCLUSION: Puerarin down-regulates the expression of FKN and suppresses the development of pulmonary hypertension and pulmonary vessel remodeling.     

Changes of hypoxia inducible factor 1 alpha and heme oxygenase 1 in the process of hypoxic pulmonary hypertension development in rats

AIM: To observe the expression of hypoxia inducible factor-1α (HIF-1α) gene and heme oxygenase-1 (HO-1) gene in pulmonary arteries in hypoxic rats. METHODS: Forty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary pressure (mPAP), vessel morphometry, right ventricle hypertrophy index (RVHI) were measured. Lungs were either inflation fixed for immunohistochemistry and in situ hybridization or frozen for later measurement of HO-1 enzyme activity. RESULTS: During hypoxia, mPAP increased to significantly higher values than the control values after 7-day of hypoxia，reaching its peak after 14-day of hypoxia, then remained on the high level. Pulmonary artery remodeling developed significantly after 14-day of hypoxia. Expression of HIF-1α protein in control was poorly positive, but was up-regulated in pulmonary arterial tunica intimae of all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein were markedly up-regulated after 3-day and 7-day of hypoxia, then tended to decline after 14-day and 21-day of hypoxia. HIF-1α mRNA staining was poorly positive in control, hypoxia for 3 days and hypoxia for 7 days, but began to enhance significantly after 14-day of hypoxia, then remained stable. Expression of HO-1 protein began to increase after 7-day of hypoxia, reaching its peak after 14-day of hypoxia, then remained stable. Expression of HO-1 mRNA began to increase after 3-day of hypoxia, reaching its peak after 7-day of hypoxia, then declined. CONCLUSION: HIF-1α and HO-1 are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats. Furthermore, HIF-1α may inter-regulate with HO-1 gene in this process.     

Effect of Panax notoginoside on pulmonary arterial pressure and expression of p38 MAPK in lung tissues of hypoxic rats

AIM: To observe the effect of Panax notoginoside (PNS) on the pulmonary artery pressure and the p38 mitogen-activated protein kinase(p38 MAPK) in lung tissues of rats treated with hypoxia. METHODS: Thirty adult male SD rats were randomly divided into 3 groups. The rats in normal control group were exposed to normal conditions, the rats in hypoxia group were exposed to isobaric hypoxia, and the rats in hypoxia+PNS group were treated with PNS under the condition of hypoxia. After 4 weeks of treatment, the mean pulmonary arterial pressure (mPAP) and the mean carotid arterial pressure (mCAP) were measured by cardiac catheterization. The heart was isolated, and the right ventricle (RV), left ventricle plus ventricular septum (LV+S) were weighed to calculate the ratio of RV/(LV+S).The quantity of phospho-p38 MAPK(p-p38 MAPK) in rat pulmonary arterioles was determined by the method of immunohistochemistry and the mRNA content of p38 MAPK was tested by RT-PCR. RESULTS: The mPAP and RV/(LV+S) in hypoxia group were higher than those in normal control group. The expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues were higher than those in normal control group (P<0.05). The mPAP, RV/(LV+S), the expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues in hypoxia+PNS group were significantly lower than those in hypoxia group (P<0.05).CONCLUSION: PNS possesses the preventive and therapeutic effect on hypoxic pulmonary hypertension by decreasing p-p38 MAPK and down-regulation of p38 MAPK mRNA in the lungs.     

Effects of adipose tissue-derived mesenchymal stem cells on calcium channels of pulmonary artery in rats with pulmonary arterial hypertension induced by monocrotaline

AIM: To investigate the effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) on calcium channels of pulmonary artery in monocrotaline (MCT)-induced pulmonary hypertensive rats.METHODS: ADMSCs were isolated from adipose tissue by collagenase digestion. Twenty-four Sprague-Dawley rats were randomly divided into 3 groups: normal control (Ctr) group, pulmonary arterial hypertension (PAH) group and ADMSCs transplantation group. Mean pulmonary arterial pressure (MPAP) was measured by catheterization, and right ventricular hypertrophy index (RVHI) was calculated. The expression of voltage-gated calcium channel α1c subunit (CaVα1c), sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA-2a), inositol 1,4,5-triphosphate receptor 1(IP3R-1), transient receptor potential channel 1 (TRPC1) and TRPC6 at mRNA and protein levels in the pulmonary trunks was determined by RT-PCR and Western blotting, respectively.RESULTS: MPAP and RVHI were higher in PAH group than those in Ctr group, while those in ADMSCs group were significantly decreased as compared with PAH group. The expression of CaVα1c, TRPC1 and TRPC6 at mRNA and protein levels was obviously increased in PAH group as compared with Ctr group, while that in ADMSCs group was significantly decreased as compared with PAH group. Compared with Ctr group, the expression of SERCA-2a and IP3R-1 at mRNA and protein levels was obviously decreased in PAH group, while that in ADMSCs group was significantly increased as compared with PAH group.CONCLUSION: MPAP and RVHI are attenuated by ADMSCs in MCT-induced pulmonary hypertensive rats. The reduction of pulmonary arterial pressure by ADMSCs transplantation in MCT-induced pulmonary hypertensive rats may be related to the changes of calcium channels.     

Baicalein inhibits monocrotaline-induced vascular wall thickening in rats with pulmonary hypertension

AIM:To investigate the effects of baicalein on pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) in rats, and its molecular mechanism was further explored. METHODS:Male SD rats (n=28) were randomly divided into 4 groups:control group, MCT group, MCT+baicalein 50 mg/kg group and MCT+baicalein 100 mg/kg group. The PAH model was established by subcutaneous injection of MCT. After 2 weeks of modeling, the rats in baicalein treatment groups were gavaged baicalein 50 and 100 mg·kg^-1·d^-1 for 14 d, the rats in control group were administered with saline. After 4 weeks of modeling, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) were detected. Masson staining was used to detect the degree of lung fibrosis. The pathomorphological changes of the pulmonary vessels were observed by HE staining. Western blot was used to detect the expression of α-smooth muscle actin (α-SMA) in the lung tissue and the phosphorylation p38, ERK and JNK in the artery. RESULTS:Compared with the control group, RVSP, RVHI and RVMI increased significantly in the MCT group (P<0.01). Pulmonary fibrosis and the thickening of pulmonary artery wall were observed. α-SMA was up-regulated and p38, ERK and JNK was activated significantly (P<0.01). Compared with the MCT group, baicalein (50 and 100 mg/kg) significantly decreased the RVSP, RVHI and RVMI (P<0.01). Lung fibrosis was reduced and the vascular wall thickening was decreased in baicalein-treated groups. Baicalein (50 and 100 mg/kg) inhibited the phosphorylation of p38, ERK and JNK compared with the MCT group (P<0.01). CONCLUSION:Baicalein ameliorates MCT-induced PAH by the inhibition of pulmonary artery wall thickening at least partially via MAPK signaling pathway.     

Expression and distribution of osteopontin in the development of pulmonary hypertension induced by hypoxia-hypercapnia

AIM: To study the expression and distribution of osteopontin (OPN) in lungs and pulmonary arteries in pulmonary hypertensive rats induced by hypoxia-hypercapnia, and to explore the role of OPN in pathogenesis of pulmonary hypertension. METHODS: Forty-eight male Sprague-Dawley rats (Weight 180 g-220 g) were randomly divided into four groups: normal control group (NC), hypoxic hypercapnia 1-week，2-week and 4-week group (1HH, 2HH and 4HH). The expressions of OPN mRNA and protein in lungs and pulmonary arteries were detected by RT-PCR and immunohistochemistry. ELISA was used to detect the concentration of OPN in lung homogenates. The content of OPN in pulmonary arteries was detected by Western blotting. RESULTS: ① The mean pulmonary artery pressure (mPAP) and weight ratio of right ventricle to left ventricle and septum ［RV/(LV+S)］ in all hypoxic hypercapniac groups were higher than those in normal control group (P<0.01), respectively. Differences of mean carotid artery pressure (mCAP) among these four groups were not significant (P>0.05). ② The expression of OPN mRNA was significantly increased in pulmonary arteries and lung tissues in hypoxic hypercapnic groups compared with normal control group (P<0.01). ③ The result of immunohistochemistry showed that OPN was only detected in bronchus and alveolar epithelium, but not detected in pulmonary arterioles of normal control group. In contrast，OPN expression was evident in pulmonary arterioles of 1HH rats，especially in media. Moreover, the expression of OPN was markedly increased in group 2HH and 4HH. ④ OPN levels in lung homogenates in 1HH, 2HH and 4HH were increased by 69%, 128% and 187% (P<0.01), respectively, compared with control rats. ⑤ Western blotting analysis showed that the contents of OPN were significantly higher in all hypoxic hypercapnic groups than those in NC group (P<0.01).CONCLUSION: The expressions of OPN in pulmonary arteioles and lung are increased in rats with pulmonary hypertension. OPN might play an important role in the pathogenesis of pulmonary hypertension induced by chronic hypoxia and hypercapnia.     

Effect of diltiazem on pulmonary arterial pressure and ceNOS mRNA expression in pulmonary arteries in chronic hypoxic hypercapnic rats

AIM: To investigate the effect of diltiazem on mean pulmonary arterial pressure (mPAP) and nitric oxide synthase (NOS) in arterioles in chronic hypoxic hypercapnic rats. METHODS: Twenty-four rats were randomly divided into three groups: control group (A), hypoxic hypercapnic group (B), hypoxic hypercapnia+ diltiazem group (C), constitutive endothelial NOS (ceNOS) were observed in arterioles of rats using the technique of immunohistochemistry, ceNOS mRNA were observed by the technique of in situ hybridization. RESULTS: (1) mPAP was significantly higher in rats of B group than that of A and C group(P<0.01). Differences of mCAP were not significant between A group and B groups (P>0.05), but mCAP was lower in rats of C group than that in B group. (2) Light microscopy showed WA/TA (vessel wall area/total area) was significantly lower in rats of C group than that of B group (P<0.01), electron microscopy showed that diltiazem inhibited the proliferation of smooth muscle cells and collageous fibers of pulmonary arterioles in chronic hypoxic hypercapnic rats. (3) Immunohistochemistry showed the average value of integral light density (LD) of ceNOS in pulmonary arterioles was significantly higher in rats of C group than that of B group (P<0.01), in situ hybridization showed LD of ceNOS mRNA in pulmonary arterioles was significantly higher in rats of C group than that of B group (P<0.01). CONCLUSION: Diltiazem inhibited pulmonary hypertension, the proliferation of smooth muscle cells and collagenous fibers of pulmonary arterioles in chronic hypoxic hypercapnic rats by incresing the expression of ceNOS in pulmonary arterioles.     

Effects of monocrotaline-induced pulmonary hypertension on expression of Hippo signaling pathway-related molecules in rat lung

AIM:To investigate the expression of Hippo signaling pathway-related molecules in the lung tissues of the rats with pulmonary hypertension induced by monocrotaline for exploring the significance of Hippo signaling pathway in the development of pulmonary hypertension. METHODS:SD rats (n=45) were randomly divided into control group (n=15) and model group (n=30). The rats in model group was given neck subcutaneous injection of monocrota-line at 60 mg/kg to establish pulmonary hypertension model, and the rats in control group was injected with the same volume of normal saline. Four weeks later, right ventricular systolic pressure (RVSP) was measured by right cardiac catheterization, and right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) were calculated. The remodeling of the pulmonary arterioles was observed by HE staining, and medial thickness/external diameter (M/E%) was evaluated. The fibrosis of lung tissues was detected by Masson staining. The protein expression of Yes-associated protein (YAP), tafazzin (TAZ) and TEAD was detected by immunohistochemistry, and the protein and mRNA levels of YAP, TAZ and TEAD in lung tissues were determined by Western blot and RT-qPCR. RESULTS:Compared with control group, the vascular wall in model group was thickened significantly, the M/E% was increased (P<0.01), the pulmonary fibrosis was obvious, and the RVSP and RVHI in model group were significantly higher than those in control group (P<0.01). The immunohistochemical staining showed that the protein expression of YAP, TAZ and TEAD in the pulmonary arterioles in model group was significantly higher than that in control group. The YAP, TAZ and TEAD protein and mRNA levels in the lung tissues were also higher than those in control group (P<0.05). CONCLUSION:The activation of Hippo signaling molecules may promote the remodeling of pulmonary arterioles and further regulate the development of monocrotaline-induced pulmonary hypertension.     

“中国园艺学会第七届青年学术讨论会”

中国园艺学会第九届第8次常务理事扩大会决定，“中国园艺学会第七届青年学术讨论会”由山东农业大学园艺科学与工程学院和山东省园艺学会承办，将于2006年7月或8月在山东泰安举行。     

Impact of hydrogen sulfide donor on endothelin-1 and connective tissue growth factor expression in rats with pulmonary hypertension induced by high pulmonary blood flow

AIM: To explore the possible impact of hydrogen sulfide (H2S) donor-sodium hydrosulfide (NaHS) on endothelin-1 (ET-1) and connective tissue growth factor (CTGF) expressions in rats with pulmonary hypertension induced by high pulmonary blood flow. METHODS: Thirty-two male SD rats were randomly divided into 4 groups: shunt group, shunt+NaHS group, sham group and sham+NaHS group. Rats in shunt group and shunt+NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. After 11 weeks of experiment, rat systolic pulmonary artery pressure (SPAP), lung tissue H2S, plasma ET-1 concentration and lung tissue ET-1mRNA expression, as well as pulmonary artery CTGF protein expression were detected.RESULTS: After 11 weeks of experiment, SPAP, lung tissue ET-1mRNA, plasma ET-1 as well as pulmonary artery CTGF expressions were increased markedly, respectively, whereas H2S in lung tissue decreased significantly in rats of shunt group as compared with that in sham group (all P<0.05). After administration of NaHS for 11 weeks, H2S in lung tissue increased significantly, whereas SPAP, plasma ET-1 and lung tissue ET-1 mRNA expression as well as pulmonary artery CTGF protein expression decreased significantly, respectively, in rats of shunt+NaHS group as compared with that in shunt group (all P<0.05).CONCLUSION: NaHS might be involved in the development of pulmonary hypertension induced by high pulmonary blood flow by down-regulating vasoactive peptides ET-1 and CTGF expressions in lung tissues of rats.     

Effect of hydroxylamine on pulmonary arterial hypertension induced by chronic hypoxic hypercapnia in rats

AIM: To explore the effects of hydroxylamine on the pulmonary arterial pressure in chronic hypoxic hypercapnic rats. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into 3 groups (8 rats in each group): the normal control group (NC), hypoxic hypercapnia+normal saline group (NS), hypoxic hypercapnia+hydroxylamine group (HA). The animals in NS and HA groups were kept in the O₂ (9%-11%) and CO₂ (5%-6%) cabin, 8 h a day and 6 days a week for 4 weeks. Before entering the cabin, the rats in HA group were administered with 1 mL hydroxylamine (12.5 mg/kg) by intraperitoneal injection, while the rats in NS group were given intraperitoneal injection of 1 mL saline solution. The mean pulmonary arterial pressure (mPAP) was measured by external jugular vein cannulation. The heart was removed, and the right ventricle (RV) and the left ventricle plus the septum (LV+S) were dissected. The ratio of the wet weight of the RV to that of the LV+S was calculated. The changes of the pulmonary vascular construction were observed under optical microscope. The concentration of H₂S in the plasma was measured with a spectrometer. The expression of cystathionine-γ-lyase (CSE) in the pulmonary arterioles and bronchi was measured by immunohistochemistry and RT-PCR. RESULTS: The values of mPAP, RV/(LV+S),vessel wall area/total area (WA/TA) and media thickness of pulmonary arterioles (PAMT) in NS group and HA group were significantly higher than those in NC group (P<0.05). The level of H₂S in the plasma, the content of CSE protein and the expression of CSE mRNA in NC group were significantly lower than those in NS group (P<0.05). The values of mPAP, RV/(LV+S), WA/TA and PAMT in HA group were significantly lower than those in NS group (P<0.05). The level of H₂S in the plasma, the content of CSE protein and the expression of CSE mRNA in HA group were significantly higher than those in NS group (P<0.05). CONCLUSION: Hydroxylamine may decrease the pulmonary arterial hypertension induced by chronic hypoxic hypercapnia in rats by increasing the level of H₂S in the plasma, the content of CSE protein and the mRNA expression of CSE, thus improving the pulmonary vascular structural remodeling.     

Effects of A2a adenosine receptor on hypoxic pulmonary hypertension in rats treated with salidroside

AIM: To study the protective effect of A_2a adenosine receptor (A_2aAR) on hypoxic pulmonary hypertension in the rats treated with salidroside. METHODS: Sprague-Dawley rats were randomly divided into 6 groups: normal control group, hypoxia group, hypoxia+salidroside (low dose) group, hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group, and hypoxia+CGS-21680 (a selective agonist of A_2aAR) group. Pulmonary hypertension in the rats was produced for 4 weeks. Mean pulmonary artery pressure (mPAP), mean carotid arterial pressure (mCAP) and the weight ratio of right ventricle/(left ventricle+septum)［RV/(LV+S)］ were measured. The expression of A_2aAR in the pulmonary arterioles was determined by immunohistochemistry and in situ hybridization. The mRNA expression of A2aAR in the lung tissues was detected by real-time RT-PCR. The protein level of A_2aAR in the lung tissues was analyzed by Western blotting. RESULTS: The mPAP in hypoxia group was significantly higher than that in normal control group. The mPAP in hypoxia+salidroside (high dose) group and CGS-21680 group was significantly lower than that in hypoxia group. RV/(LV+S) in hypoxia group were significantly higher than that in normal control group. RV/(LV+S) in hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group and CGS-21680 group were lower than that in hypoxia group. The ratio of vessel wall area/vessel total area (WA/TA) in hypoxia group was significantly higher than that in normal control group. WA/TA in hypoxia+salidroside (low dose) group, hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group and CGS21680 group were obviously lower than that in hypoxia group. The expression of A_2aAR was significantly higher in hypoxia group than that in normal control group. The expression of A_2aAR in hypoxia+salidroside (high dose) group and CGS-21680 group was obviously higher than that in hypoxia group. CONCLUSION: The A_2aAR attenuates pulmonary vessel remodeling and pulmonary hypertension induced by hypoxia. Salidroside protects the pulmonary vessel from remodeling and inhibits the development of hypoxia-induced pulmonary hypertension by up-regulation of A_2aAR expression.     

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.     

Intermedin inhibits pulmonary collagen synthesis in rats with pulmonary hypertension induced by high pulmonary blood flow

AIM: To explore the regulatory effect of intermedin (IMD) on pulmonary collagen synthesis and accumulation in rats with pulmonary hypertension induced by high pulmonary blood flow.METHODS: Healthy male SD rats (n=20) were randomly divided into control group (n=7), shunt group (n=7) and shunt with IMD group (n=6). The shunting of abdominal aorta and inferior vena cava was produced in rats of shunt group and shunt with IMD group. After 8 weeks, IMD was administered into the rats of shunt with IMD group subcutaneously by mini-osmotic pump for 2 weeks. Mean pulmonary artery pressure (mPAP), relative medial thickness (RMT) of pulmonary arteries, contents of hydroxyproline, collagen type I and III, bone morphogenetic protein-2 (BMP-2), and the mRNA expression of procollagen I and III in lung tissues were measured and compared. RESULTS: Compared with control group, mPAP and RMT of medium and small pulmonary arteries in the rats of shunt group were significantly increased. Meanwhile, the lung hydroxyproline, collagens I and III and BMP-2 contents, and the mRNA expression of lung procollagen I and III were all significantly increased compared with control group. However, IMD significantly decreased mPAP, alleviated the changes of pulmonary vascular micro-structure, decreased the collagen accumulation and pulmonary tissue homogenate BMP-2 contents, and inhibited the mRNA expression of procollagen I and III in the lung tissue of shunting rats.CONCLUSION: IMD plays a protective role in the development of pulmonary hypertension and pulmonary vascular structural remodeling induced by high blood flow by inhibiting pulmonary collagen synthesis and accumulation, possibly in association with the BMP-2 pathway.     

Expression of Jagged2/Notch3 signaling molecules in pulmonary hypertensive rats induced by monocrotaline

AIM: To study the expression of Jagged2/Notch3 signaling molecules in pulmonary vascular wall of pulmonary hypertensive rats induced by monocrotaline. METHODS: SD rats were randomly divided into normal control group (C group,n=15), solvent control group (S group,n=15) and monocrotaline model groups (M group,n=15). The model of pulmonary hypertension was established by a single subcutaneous injection of monocrotaline (50 mg/kg). The rats in S group were given a single subcutaneous injection of the same dose of solvent. After 4 weeks, the pulmonary vascular remodeling was assessed by HE staining, and the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) were determined by right heart catheterization. The expression of Jagged2/Notch3/Hes5 molecules in the pulmonary vascular wall was detected by immunohistochemical method and real-time PCR. RESULTS: Compared with S group and C group, the percentage of medial wall thickness of smaller arteries in model group increased significantly (P<0.01). The levels of mPAP and RVSP in M group were significantly higher than those in S group and C groups (P<0.01). The results of real-time PCR showed that the expression of Jagged2, Notch3 and Hes5 was significantly increased in M group compared with S group and C group. The data from immunohistochemical detection indicated that Jagged2 mainly expressed in the intima of small lung artery, Notch3 and Hes5 mainly expressed in the medial smooth muscle cells. Compared with S group and C group, the expression of Jagged2 and Notch3 was significantly increased in the lung small arteries of M group. CONCLUSION: The activation of Jagged2/Notch3 signaling pathway might play an important role in the formation of pulmonary hypertension.