OBJECTIVEPulmonary hypertension (PH) is a common complication of congenital heart defects with a left-to-right shunt characterized by high pulmonary blood flow. Pulmonary vascular structural remodeling (PVSR) is the pathological basis of PH. However, the pathophysiologic features and mechanisms responsible for PH and PVSR induced by increased pulmonary blood flow have not been fully understood. The present study was designed to explore the possible effect and mechanism of hydrogen sulfide (H(2)S) on the regulation of PVSR induced by high pulmonary flow in rats.

METHODSThirty-two male SD rats, weighing 120 - 140 g, were randomly divided into shunt group (n = 8), shunt + NaHS group (n = 8), control group (n = 8) and control + NaHS group (n = 8). 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. Rats in the control and control + NaHS groups underwent the same experimental protocol as mentioned above except for the shunt procedure. Rats in the shunt + NaHS and control + NaHS groups were intraperitoneally injected with NaHS at 56 micromol/(kgxd), and rats in the shunt and control groups were injected with the same volume of physiological saline. After 11 weeks of experiment, rats were sacrificed and lung tissues were obtained. The percentage of muscularized artery (MA) was calculated. The changes in relative medial thickness (RMT) in small pulmonary arteries and median pulmonary arteries were examined. Proliferative cell nuclear antigen (PCNA), extracellular signal-regulated kinase (ERK1) and phosphorylation extracellular signal-regulated kinase (P-ERK1) protein expression were examined by Western blot, and at the same time, PCNA protein expression by pulmonary artery smooth muscle cells was observed by immunohistochemistry.

RESULTSAfter 11 weeks of shunt, compared with control group, the percentage of MA increased significantly (25.12 +/- 2.26 vs 14.42 +/- 3.41, P < 0.05), and RMT in small pulmonary arteries and median pulmonary arteries increased significantly in rats of shunt group (23.6 +/- 3.5 vs 12.6 +/- 2.1, 24.8 +/- 1.9 vs 13.5 +/- 2.2, P < 0.05 for all). PCNA protein expression in small and median pulmonary arteries increased significantly (0.49 +/- 0.04 vs 0.39 +/- 0.07, 0.46 +/- 0.08 vs 0.36 +/- 0.05, P < 0.01 for all), and the ratio of PERK/ERK1 protein expression of pulmonary arteries increased significantly (P < 0.01) in rats of shunt group compared with those of control group. After the administration of exogenous H(2)S donor, NaHS, for 11 weeks, in contrast to rats in shunt group, the percentage of MA decreased significantly (21.5 +/- 2.0 vs 25.1 +/- 2.3, P < 0.05), and RMT in small and median pulmonary arteries decreased significantly (20.2 +/- 2.8 vs 23.6 +/- 3.5, 20.8 +/- 3.1 vs 20.8 +/- 3.1, P < 0.05 for all) in rats of shunt + NaHS group. PCNA protein expression in small and median pulmonary artery smooth muscle cells decreased significantly (0.32 +/- 0.06 vs 0.49 +/- 0.04, 0.29 +/- 0.07 vs 0.46 +/- 0.08, P < 0.01 for all), and the ratio of PERK/ERK1 protein expression of pulmonary arteries decreased significantly (P < 0.01) in rats of shunt + NaHS group compared with that of shunt group.

CONCLUSIONH(2)S may play a regulatory role in pulmonary vascular structural remodeling induced by high pulmonary blood flow via mitogen-activated protein kinase (MAPK)/ERK signal transduction pathway.

Animals ; Hydrogen Sulfide ; pharmacology ; Hypertension, Pulmonary ; pathology ; physiopathology ; Lung ; pathology ; Male ; Pulmonary Artery ; drug effects ; physiopathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVEHypoxic pulmonary hypertension is an important pathophysiologic process of various cardiovascular diseases. Sulfur dioxide (SO2) was considered as a kind of toxic gas previously, but recent studies suggested that SO2 could act as a key bioactive molecule in the pathogenesis of cardiovascular diseases. Therefore, this study was designed to examine the effect of sulfur dioxide on pulmonary vascular structure of hypoxic pulmonary hypertensive rats treated with SO2 donor substances.

METHODSThe rats were randomly divided into 3 groups: control group(n = 8), hypoxic group(n = 8) and hypoxic + SO2 group (n = 10, treated with SO2 donor Na2SO3/NaHSO3). The rats of hypoxic group and hypoxic + SO2 group were under a hypoxic condition for 21 days, while the rats of control group were exposed to room air. The mean pulmonary artery pressure was tested by means of right cardiac catheterization and the content of SO2 in plasma was investigated by high performance liquid chromatography (HPLC). The change in relative medial thickness (RMT) of pulmonary arteries was examined under optical microscope. The ultra-structural changes were observed under a transmission electron microscope. The data were analyzed through one-way analysis of variance (ANOVA) by SPSS 13.0 software.

RESULTSCompared with control group [(2.25 +/- 0.50) kPa], the mean pulmonary artery pressure of hypoxic group [(5.12 +/- 0.51) kPa] raised significantly (t = 5.091, P < 0.01) and RMT of hypoxic group (9.66 +/- 1.27) compared with control group (6.83 +/- 1.57) significantly raised (t = 3.392, P < 0.01). Ultrastructural observation showed the proliferation and degeneration of endothelial cells in small pulmonary arteries in rats with pulmonary hypertension. The internal elastic lamina was irregular. The proliferation of medial smooth muscle cells of arteries was shown at the level of respiratory bronchioles. The collagens also increased. Meanwhile, compared with control group [(33.36 +/- 5.62) micromol/L], the content of SO2 in plasma of hypoxic group [(27.01 +/- 4.17) micromol/L] declined (t = 2.067, P < 0.05). Whereas compared with that of hypoxic group [(5.12 +/- 0.51) kPa], the mean pulmonary artery pressure of hypoxic + SO2 group [(3.94 +/- 0.33) kPa] declined (t = 2.712, P < 0.01) and RMT of hypoxic + SO2 group (6.97 +/- 1.83) decreased compared with hypoxic group (9.66 +/- 1.27) (t = 3.009, P < 0.01). Compared with those of hypoxic group, the pulmonary artery ultrastructural changes in hypoxic group ameliorated obviously after using exogenous sulfur dioxide donor. The endothelial cells became flat and the smooth muscle cells of arteries slightly enlarged and arranged regularly. At the same time, compared with hypoxic group [(27.01 +/- 4.17) micromol/L], the content of SO2 in plasma of hypoxic + SO2 group [(29.89 +/- 4.52) micromol/L] raised (t = 1.263, P > 0.05).

CONCLUSIONSulfur dioxide plays an important role in the regulation of small pulmonary artery structural changes in hypoxic pulmonary hypertensive rats. The hypoxic pulmonary hypertensive damages can be ameliorated significantly after using exogenous SO2 donor.

Animals ; Hypertension, Pulmonary ; blood ; pathology ; physiopathology ; Hypoxia ; blood ; pathology ; physiopathology ; Male ; Pulmonary Artery ; drug effects ; pathology ; Rats ; Rats, Wistar ; Sulfur Dioxide ; adverse effects ; blood

Intravenous and intratracheal implantation of mesenchymal stem cells (MSCs) may offer ameliorating effects on pulmonary hypertension (PH) induced by monocrotaline (MCT) in rats. The aim of this study was to examine the anti-remodeling effect of intravenous MSCs (VMSCs) and intratracheal MSCs (TMSCs) in rats with PH, and the underlying mechanisms. MSCs were isolated from rat bone marrow and cultured. PH was induced in rats by intraperitoneal injection of MCT. One week after MCT administration, the rats were divided into 3 groups in terms of different treatments: VMSCs group (intravenous injection of MSCs), TMSCs group (intratracheal injection of MSCs), PH group (no treatment given). Those receiving saline instead of MCT served as negative control (control group). Pulmonary arterial structure was pathologically observed, pulmonary arterial dynamics measured, and remodeling-associated cytokines Smad2 and Smad3 detected in the lungs, three weeks after MCT injection. The results showed that PH group versus control group had higher pulmonary arterial pressure (PAP) and wall thickness index (WTI) 21 days after MCT treatment. The expression of phosphorylated (p)-Smad2 and the ratio of p-Smad2/Smad2 were much higher in PH group than in control group. Fluorescence-labeled MSCs were extensively distributed in rats' lungs in VMSCs and TMSCs groups 3 and 14 days after transplantation, but not found in the media of the pulmonary artery. WTI and PAP were significantly lower in both VMSCs and TMSCs groups than in PH group three weeks after MCT injection. The p-Smad2 expression and the ratio of p-Smad2/Smad2 were obviously reduced in VMSCs and TMSCs groups as compared with those in PH group. In conclusion, both intravenous and intratracheal transplantation of MSCs can attenuate PAP and pulmonary artery remodeling in MCT-induced PH rats, which may be associated with the early suppression of Smad2 phosphorylation via paracrine pathways.
Animals ; Atrial Remodeling ; drug effects ; physiology ; Hypertension, Pulmonary ; chemically induced ; physiopathology ; Male ; Mesenchymal Stromal Cells ; pathology ; Monocrotaline ; pharmacology ; Pulmonary Artery ; drug effects ; physiopathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the expression of inducible heme oxygenase (HO-1) gene in pulmonary artery smooth muscle cells (PASMCs) exposed to hypoxia, and the influence of carbon monoxide (CO) on the proliferation of PASMCs under hypoxic conditions.

METHODSPrimary culture of rat PASMCs were passed every 3 days, and the 3 - 5 passages were used. After exposure to hypoxic conditions (95% N2, 5% CO(2)) 0, 12, 24 and 48 hours, the level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR). The volume of COHb in the medium was measured spectrophotometrically. The cyclic guanosine mono-phosphate (cGMP) concentration of cell extracts was determined by radioimmunoassay. PASMCs were divided into 5 groups, cultured under normoxia and hypoxia and treated with hemin, hemoglobin (Hb) and exogenous CO respectively. Then 3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay and immunocytochemical staining were used to study the energy metabolism and the expression of proliferating cell nuclear antigen (PCNA) in PASMCs. Flow cytometry was used to analyze the cell cycle of PASMCs.

RESULTSAfter exposure to hypoxic conditions for 12, 24, and 48 hours, the HO-1 mRNA increased by 2.7%, 5.7% and 27.1% respectively (P < 0.01). The carboxy-hemoglobin (COHb) in the medium increased by 13.8%, 31.0% and 93.1% (P < 0.01); the cGMP concentrations were 2.7, 4.0 and 6.8-fold compared with the control group (P < 0.01 and P < 0.05). In comparison with the control group, the value of MTT colorimetric assay, the immunocytochemical staining of PCNA and the percentages of PASMCs in S and G2M phases in the hypoxic group were significantly higher (P < 0.01). After treatment with Hemin and CO, the results of the above analysis decreased significantly (P < 0.01 and P < 0.05), but increased significantly after treatment with Hb (P < 0.01 and P < 0.05).

CONCLUSIONSThe expression of HO-1 gene in PASMCs is upregulated by hypoxia and the production of endogenous CO is elevated as well. The endogenous CO suppresses the proliferation of PASMC in an autocrine way. Both the induction of endogenous CO by Hemin and the treatment with exogenous CO can suppress the proliferation of rat PASMCs of under hypoxic conditions.

Carbon Monoxide ; pharmacology ; physiology ; Cell Division ; drug effects ; Cells, Cultured ; Gene Expression ; Heme Oxygenase (Decyclizing) ; genetics ; Hypoxia ; pathology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; pathology ; Pulmonary Artery ; pathology

Pulmonary arterial hypertension (PAH) secondary to chronic obstructive pulmonary disease (COPD) is incurable and it has an unpredictable survival rate. Two men who suffered from COPD presented with progressive dyspnea and edema, respectively. PAH, as estimated by the peak velocity of tricuspidal regurgitation, and the depressed myocardial performance index (MPI) of the right ventricle (RV) were noted on echocardiography. In addition to the baseline therapy for their depressed ventilatory function, we prescribed tadalafil 10 mg orally every other day for 2 weeks and then we doubled the dosage. They well tolerated the medication without any notable side effects. After 4 weeks of tadalafil treatment, the patients' pulmonary arterial pressure was decreased and the MPI of the RV was improved in both. The exercise capacity, as measured by the respiratory oxygen uptake, also improved from 10.9 mL/kg/min to 13.8 mL/kg/min in one patient. We report here on 2 patients with PAH secondary to COPD, and they showed notable improvement of their pulmonary hemodynamics and exercise capacity with the administration of tadalafil.
Pulmonary Disease, Chronic Obstructive/*complications ; Pulmonary Artery/drug effects/*pathology ; Phosphodiesterase Inhibitors/*therapeutic use ; Oxygen Consumption/drug effects ; Middle Aged ; Male ; Hypertension, Pulmonary/*drug therapy/etiology ; Humans ; Exercise Tolerance/drug effects ; Carbolines/*therapeutic use

OBJECTIVEPulmonary hypertension is a proliferative vascular disease characterized by pulmonary vascular structural remodeling. Until now, the pathogenesis of pulmonary hypertension is still not fully understood. Although considerable progress has been made, there is, to date, no cure for advanced pulmonary vascular disease. Recently, a number of studies suggest that endogenous vascular elastase (EVE) play a role in the vascular changes associated with pulmonary hypertension. The purpose of the study was to determine whether an elastase inhibitor might reverse advanced pulmonary vascular disease produced in rats by injection of monocrotaline.

METHODSOne hundred and twenty male Sprague-Dawley rats were used in this study. The rats were divided into three groups: control, model and ZD-0892 groups. In the model and ZD-0892 groups, the rats were subjected to a single subcutaneous injection of monocrotaline (60 mg/kg) in the hind flank, while the rats in control group received an equivalent volume of 0.9% saline. From day 21, the rats in the ZD-0892 and model groups received twice-daily gavage tube feedings of either ZD-0892 at a dose of 240 mg/kg per day or its administration vehicle, while the rats in control group were subjected to an equivalent volume of 0.9% saline. On days 21, 28 and 35 post-injection, the elastolytic activity was measured with a fluorescence microplate reader and pulmonary artery pressure was detected via catheterization. Meanwhile, the lungs were evaluated morphologically, using the barium-gelatin perfusion technique.

RESULTSThe injection of monocrotaline led to severe pulmonary hypertension in rats 21 days later and pulmonary artery elastolytic activity increased remarkably. A 1-week treatment with ZD-0892 resulted in declines in elastase activity. This was associated with significant declines in pulmonary artery pressure, decreases in muscularization of peripheral arteries and reductions in medial hypertrophy. After 2 weeks, elastase activity returned to normal level. Pulmonary artery pressure and structure were normalized.

CONCLUSIONIncreased elastase activity is important in the development of vascular changes and progressive pulmonary hypertension. ZD-0892 can suppress the elastase activity and completely reverse the fatal pulmonary hypertension induced by monocrotaline in rats.

Animals ; Hypertension, Pulmonary ; chemically induced ; drug therapy ; Male ; Monocrotaline ; toxicity ; Pancreatic Elastase ; antagonists & inhibitors ; Pulmonary Artery ; drug effects ; pathology ; physiopathology ; Pyrroles ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfonamides ; pharmacology

The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs) proliferation and migration induced by cigarette smoke extract (CSE). The eukaryotic expression vector of antisense cyclin D1 gene (pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCs using liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups: control group, vector group, antisense cyclin D1 group, 5% CSE group, vector+5% CSE group and antisense cyclin D1+5% CSE group. The expressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. The proliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemical staining. The migration of HPASMCs was measured by Transwell cell test. The results showed that the eukaryotic expression vector of antisense cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA and protein levels in antisense cyclin D1 group were significantly lower than those in control group (P<0.05). In 5% CSE group, the cyclin D1 mRNA and protein levels were elevated significantly compared with those in control group (P<0.05), and the indicators of cell and migration in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P<0.05). These results suggest that CSE could promote HPASMCs proliferation and migration through up-regulation of cyclin D1 expression. PIRES2-EGFP-ascyclin D1 could attenuate CSE-induced proliferation and migration of HPASMCs by suppressing the expression of cyclin D1, which implicates that cyclin D1 might be involved in the process of HPASMCs proliferation and migration stimulated by CSE.
Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; physiology ; Humans ; Muscle, Smooth, Vascular ; cytology ; pathology ; Myocytes, Smooth Muscle ; cytology ; pathology ; Pulmonary Artery ; cytology ; pathology ; Smoke ; adverse effects ; Tobacco ; adverse effects

To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether its mechanism is achieved by regulating reactive oxygen. PASMCs of primarily cultured rats (2-5 generations) were selected in the experiment. MTT, Western blot, FCM and DCFH-DA were used to observe Pue's effect the proliferation of PASMCs. The Western blot was adopted to detect whether ROS participated in Pue's effect in inhibiting PASMC proliferation. The PASMCs were divided into five groups: the normoxia group, the hypoxia group, the hypoxia + Pue group, the hypoxia + Pue + Rotenone group and the hypoxia + Rotenone group, with Rotenone as the ROS blocker. According to the results, under the conditions of normoxia, Pue had no effect on the PASMC proliferation; But, under the conditions of hypoxia, it could inhibit the PASMC proliferation; Under the conditions of normoxia and hypoxia, Pue had no effect on the expression of the tumor necrosis factor-α (TNF-α) among PASMCs, could down-regulate the expression of hypoxia-induced cell cycle protein Cyclin A and proliferative nuclear antigen (PCNA). DCFH-DA proved Pue could reverse ROS rise caused by hypoxia. Both Rotenone and Pue could inhibit the up-regulated expressions of HIF-1α, Cyclin A, PCNA caused by anoxia, with a synergistic effect. The results suggested that Pue could inhibit the hypoxia-induced PASMC proliferation. Its mechanism may be achieved by regulating ROS.
Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hypoxia ; pathology ; Isoflavones ; pharmacology ; Male ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Pulmonary Artery ; cytology ; drug effects ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

Acetophenones ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; pathology ; Pulmonary Artery ; drug effects ; pathology

AIMTo investigate the effects of hypoxia on the proliferation and apoptosis of PASMC, to evaluate the role of iNOS protein expression and ADM on the hypoxic pulmonary hypertension (HPH) pathogenesis.

METHODSTo culture rat pulmonary artery smooth muscle cell (PASMC), cultured PASMC cells were grouped into: normoxic group; hypoxic group; hypoxia + L-NAME group; hypoxia+ ADM group. Proliferation of PASMC were investigated by MTT and PCNA. Apoptosis of PASMC were examined by flow-cytometry. Westen blot was used to measure protein expression of iNOS induced by hypoxia.

RESULTS(By MTT, the value of 24 h hypoxia was significantly higher than that in the normoxic group (P < 0.01), the value of the hypoxia + ADM was significantly lower than that in hypoxia group, the value of the hypoxia + L-NAME was significantly higher than those of hypoxic group and normoxic group (P < 0.01). (2) By immunohistochemistry, PCNA was poorly positive in PASMC, whereas positive after 24 h hypoxia (P < 0.01), ADM inhibited the expression of PCNA significantly (P < 0.01), whereas L-NAME increased the expression of PCNA significantly (P < 0.01). (3) By FCM, apoptosis index was not significantly different between the normoxic group, hypoxic group, hypoxia + L-NAME and hypoxia + ADM (P > 0.05). (4) By Western blot, iNOS expression was poorly positive in control group, positive after 4 h hypoxia (P < 0.01), increasing as the hypoxia environment continued (P < 0.01). L-NAME had no effect on iNOS protein, ADM promoted iNOS expression (P < 0.01).

CONCLUSION(1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) Hypoxia induces the expression of iNOS, ADM can increase expression of iNOS, ADM and INOS plays a role of protection in HPH pathogenesis.

Adrenomedullin ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cell Proliferation ; drug effects ; Cells, Cultured ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; pathology ; Nitric Oxide Synthase Type II ; metabolism ; Pulmonary Artery ; cytology ; pathology ; Rats ; Rats, Sprague-Dawley