Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 microgram/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.
Animals ; Blood Pressure/drug effects ; Bronchi/cytology/drug effects/metabolism ; Endothelial Cells/cytology/drug effects/metabolism ; Epithelial Cells/cytology/drug effects/metabolism ; Hypertension, Pulmonary/chemically induced/*metabolism ; Immediate-Early Proteins/genetics/*metabolism ; Intercellular Signaling Peptides and Proteins/genetics/*metabolism ; Lung/cytology/drug effects/*metabolism ; Male ; Monocrotaline/*toxicity ; Pulmonary Alveoli/cytology/drug effects/metabolism ; Pulmonary Artery/cytology/drug effects/metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

Hypoxic pulmonary hypertension (HPH) is a syndrome characterized by the increase of pulmonary vascular tone and the structural remodeling of peripheral pulmonary arteries. The aim of specific therapies for hypoxic pulmonary hypertension is to reduce pulmonary vascular resistance, reverse pulmonary vascular remodeling, and thereby improving right ventricular function. Iptakalim, a lipophilic para-amino compound with a low molecular weight, has been demonstrated to be a new selective ATP-sensitive potassium (K(ATP)) channel opener via pharmacological, electrophysiological, biochemical studies, and receptor binding tests. In hypoxia-induced animal models, iptakalim decreases the elevated mean pressure in pulmonary arteries, and attenuates remodeling in the right ventricle, pulmonary arteries and airways. Furthermore, iptakalim has selective antihypertensive effects, selective vasorelaxation effects on smaller arteries, and protective effects on endothelial cells, but no effects on the central nervous, respiratory, digestive or endocrine systems at therapeutic dose. Our previous studies demonstrated that iptakalim inhibited the effects of endothelin-1, reduced the intracellular calcium concentration and inhibited the proliferation of pulmonary artery smooth muscle cells. Since iptakalim has been shown safe and effective in both experimental animal models and phase I clinical trials, it can be a potential candidate of HPH in the future.
Animals ; Antihypertensive Agents ; therapeutic use ; Calcium ; metabolism ; Disease Models, Animal ; Endothelin-1 ; metabolism ; Hypertension, Pulmonary ; drug therapy ; Hypoxia ; drug therapy ; KATP Channels ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Propylamines ; therapeutic use ; Pulmonary Artery ; drug effects

The study was designed to explore the alteration of intracellular calcium concentration ([Ca²⁺]i), induced by transient receptor potential melastatin 8 (TRPM8) channel-specific agonist menthol, in pulmonary arterial smooth muscle cells (PASMCs) between control and pulmonary hypertensive (PH) rats. PH rat models were established by means of chronic hypoxia (CH) and monocrotaline (MCT) injection, respectively. PASMCs from control and PH rats were cultured. The change of [Ca²⁺]i in PASMCs induced by menthol, and the effect of TRPM8 channel-specific antagonist BCTC on the change of [Ca²⁺]i, were observed. Cellular localization of TRPM8 was examined by using immunohistochemistry. Results showed that menthol increased [Ca²⁺]i in the control PASMCs both in Ca²⁺ -normal and Ca²⁺ - free Tyrode's solutions, and at the same time BCTC could inhibit these two kinds of elevations. Compared with the control group, elevations of [Ca²⁺]i were decreased notably in CH- and MCT-pretreated PASMCs superfused with 2 mmol/L Ca²⁺ - or 0 Ca²⁺ -Tyrode's solutions. Immunohistochemical localization experiments showed that the whole PASMCs were dyed brown except for the nucleus. This study verified that TRPM8 exists both in membrane and sarcoplasmic reticulum of PASMCs. In addition, CH- and MCT-pretreatment could independently down-regulate the Ca²⁺ influx and Ca²⁺ release mediated by TRPM8 channel.
Animals ; Calcium ; metabolism ; Cells, Cultured ; Menthol ; pharmacology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Sarcoplasmic Reticulum ; metabolism ; TRPM Cation Channels ; metabolism

AIMTo study the influence of Na+/H+ exchange inhibitor amiloride on hypoxia-induced proliferation in rats pulmonary artery smooth muscle cells (PASMCs), also observe the change of Na+/H+ exchanger-1 (NHE-1) activity and expression.

METHODSRats PASMGs were cultured in normoxia (21% O2) or hypoxia (2%O2) for 24 hours, as well as administered amiloride with various concentrations, cultured for 24 hours, then determined MTT OD values and rates of PCNA positive cells to investigate cells proliferation, moreover intracellular pH was determined by interactive Laser Cytometer, and Na+/H+ exchanger-1 mRNA expression was determined by RT-PCR.

RESULTSHypoxic exposure heightened intracellular pH and mRNA expression of NHE-1 in PASMCs, however, 3.123-50 micromol/L amiloride depressed them gradually. Additionally, hypoxic exposure raised MTT OD value and rates of PCNA positive cells, similarly, the above two indexes descended gradually with presence of 3.125-50 micromol/L amiloride.

CONCLUSIONNa+/H+ exchange inhibitor amiloride can suppress hypoxia-induced proliferation in pulmonary artery smooth muscle cells, which is due to depress activity and expression of NHE-1.

Amiloride ; pharmacology ; Animals ; Cell Hypoxia ; Cell Proliferation ; drug effects ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Pulmonary Artery ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sodium Channel Blockers ; pharmacology ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; metabolism

OBJECTIVETo study the effects of puerarin on proliferation, apoptosis and Kv1.5 gene expression of rat pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia.

METHODSThe rat PASMCs were divided into 5 groups: control group, hypoxia group, hypoxia plus puerarin (1 × 10(-5) mol/L) group, hypoxia plus puerarin (1 × 10(-4) mol/L) group and hypoxia plus puerarin (1 × 10(-3) mol/L) group, and cultured at 37°C for 24 h. The proliferation of rat PASMCs was detected by CCK-8 assay and flow cytometry, the activity of caspase-3 was measured with spectrophotometric method, Kv1.5 protein was detected by western blot, Kv1.5 mRNA was detected by real-time PCR.

RESULTSThe cell viability and proportion of synthesis phase in control group were 0.940 ± 0.045 and 9.67% ± 1.28%, which were significantly lower than those (1.296 ± 0.034 and 18.19% ± 1.19%) in hypoxia group (P < 0.05). The Caspase-3 activity, Kv 1.5 protein and Kv 1.5 mRNA in control group were 0.1073 ± 0.0113, 0.886 ± 0.038 and 0.0377 ± 0.0031, which were significantly higher than those (0.0664 ± 0.0049, 0.602 ± 0.064 and 0.0108 ± 0.0014) in hypoxia group (P < 0.05). As compared with hypoxia group, the cell viability and proportion of synthesis phase in 3 hypoxia plus puerarin groups significantly decreased, and the Caspase-3 activity, Kv 1.5 protein and Kv 1.5 mRNA in 3 hypoxia plus puerarin groups significantly enhanced (P < 0.05).

CONCLUSIONPuerarin could decrease the proliferation and increase the apoptosis induced by hypoxia in rat PASMCs, and the up-regulated expression of Kv1.5 gene may be the mechanism of puerarin effects.

Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cell Proliferation ; drug effects ; Cells, Cultured ; Isoflavones ; pharmacology ; Kv1.5 Potassium Channel ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Pulmonary Artery ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of autophagy inhibitor chloroquine (CQ) in the proliferation of pulmonary arterial smooth muscle cells (PASMCs) in hypoxia conditions.

METHODSThe following groups in this study were set up: control group, hypoxia group, 50 micromol/L CQ + hypoxia group, 50 micromol/L CQ group. The viability of PASMCs in every group was detected by MTT assay. Autophagic vacuoles in the cells were observed by MDC staining. Protein expression of microtubule associated protein light chain 3 (LC3) was measured by Western blot. Migration of PASMCs was detected by wound healing assay.

RESULTSCompared with control group, no effect on the viability of PASMCs was observed treated by CQ alone. In 1% hypoxia group, cell viability increased significantly compared with that in control group. The number of autophagic vacuoles and the rate of cell migration and also protein expression of LC3-II were also markedly increased. Compared with hypoxia group, addition of CQ increased the number of autophagic vacuoles and the levels of LC3-II protein, but decreased the proliferation and migration of PASMCs.

CONCLUSIONHypoxia could activates autophagy and contributes to proliferation and migration of PASMCs, and autophagy inhibitor CQ could decrease the effect of hypoxia on PASMCs through inhibiting autophagy process.

Autophagy ; drug effects ; Cell Hypoxia ; Cell Movement ; Cell Survival ; Cells, Cultured ; Chloroquine ; pharmacology ; Humans ; Microtubule-Associated Proteins ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; Pulmonary Artery ; cytology

OBJECTIVE: To synthesize 3, 5, 4' -trimethoxystilbene (TMS) by methylation of resveratrol (Res), a natural compound extracted from polygonum cuspidatum, to identify the chemical structure of TMS, to test its pharmacokinetics, and to determine the effects of TMS on the growth inhibition and apoptosis in pulmonary artery smooth muscle cells (PASMCs). METHODS: The chemical structure of TMS was analyzed by UV- and IR- absorption spectrometry, (1)H-NMR and (13)C-NMR spectroscopy and mass spectrometry. We measured the bioavailability, the characteristics of intestinal absorption, and the distribution of TMS in body and excretions of SD rats after oral administration of TMS. The acute toxicity of TMS in mice was tested. PASMCs were prepared from pulmonary artery of SD rats. The PASMCs were divided into 8 groups. Group of A (control) was cultured without TNF-α, TMS, or Res. Group of B (TNF-α) was cultured with 100 pg/mL TNF-α. Groups of C-E (low-high concentrations of TMS) were cultured with 100 pg/mL TNF-α and 5, 10, 20 μmol/L TMS, respectively. Groups of F-H (low-high concentrations of Res) were cultured with 100 pg/mL TNF-α and 50, 100, 200 μmol/L Res, respectively. The proliferation of PASMCs after treatment was determined by MTT assay. The apoptosis of PASMCs after treatment was determined by flow cytometry. RESULTS: The UV absorption map of TMS showed λmax(MeOH) at 318, 306.2, and 217.8 nm. Analysis of infrared spectrum of TMS showed IRvKBr max /cm at 2999, 2935, 2836, 1591, 1511 and 1456/cm. The (1)H-NMR map showed that the synthetic product contained three hydroxy groups, while (13)C-NMR map showed 17 carbon signals and some symmetrical structural fragments. Electospray ionization mass spectrometry of the productshowed m/z peaks corresponded to 271[M+H](+), 256[M+H-CH(3)](+) and 241[256-CH(3)](+); the implied relative molecular weight is 270 and the implied molecular formula is C17H18O3. These data confirm the product is 3,5,4' - trimethoxystilbene. The absolute bioavailability of TMS was 45.4%. TMS was well absorped in the upper small intestine; it was excreted in stool and bile and distributed into several tissues. The maximal tolerance dose (MTD) of TMS was 5.85 g/kg. MTT assay showed TMS inhibited the proliferation of PASMCs in a dose-dependent manner. The extent of growth inhibition in A-H groups were (4.07±2.12)%, (6.54±4.78)%, (9.35±4.26)%, (16.75±5.34)%, (23.74±7.07)%, (6.78±5.58) %, (8.81±5.16) %, and (17.81±6.03) %, respectively. Flow cytometry showed the extent of apoptosis in PASMCs (after being treated with TMS for 24 h) was significantly higher than that in PASMCs treated only with TNF-α. The apoptosis rates of A-H groups were (2.63±0.74)%, (3.54±0.81)%, (5.77±4.62)%, (11.68±5.35)%, (18.79±4.15)%, (4.11±3.59)%, (6.33±4.8) %, and (12.47±5.06)%, respectively. CONCLUSION We have confirmed our synthetic product as 3,5,4'-trimethoxystilbene (TMS), with the molecular formula of C17H18O3 and appropriate molecular weight and absorbption and NMR spectra. The bioavailability of TMS was to 45%. It strongly inhibits the proliferation of PASMCs in a dose-dependent manner and induces apoptosis of PASMCs.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; Cells, Cultured ; Male ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Pulmonary Artery ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Resveratrol ; Stilbenes ; chemical synthesis ; chemistry ; isolation & purification ; pharmacokinetics ; pharmacology

This paper is to report the exploration of the activation of Rho/ROCK signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-Nis on this pathway. PASMCs were cultured with the explant technique. MTT assay was used to explore the proliferation of PASMCs after 5-HT treated for different time and the intervening effect of m-Nis. RT-PCR and Western blot were used respectively to explore the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 in 5-HT-treated PASMCs and intervening effect of m-Nis. The results of MTT assay suggested that 5-HT (1 µmol · L(-1)) treatment for 12-72 h significantly induced the proliferation of rat PASMCs (P<0.05 or P < 0.01), which were inhibited by m-Nis (1 x 10(-5), 1 x 10(-6), l x 10(-7), 1 x10(-8) mol · L(-1)) in dose-dependent manners (P < 0.05 or P < 0.01). Similarly, the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 were also inhibited by m-Nis in different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Rho/ROCK pathway played an important role in 5-HT-induced proliferation of rat PASMCs, m-Nis inhibited 5-HT-induced proliferation obviously, which may be related to the blockage of Rho/ROCK signal pathway.
Animals ; Cell Proliferation ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Nisoldipine ; pharmacology ; Protein Phosphatase 1 ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Serotonin ; pharmacology ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVETo investigate the inhibitory effect of Yifei Huoxue Granule (, YFHXG) on the hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and its mechanism of decreasing pulmonary arterial pressure.

METHODSTwenty male Sprague-Dawley (SD) rats were randomly divided into four groups: saline, and 0.66, 3.30 and 16.50 g/kg of YFHXG groups, the saline and different concentrations of YFHXG were given twice daily for 7 days, respectively. Serum-pharmacology method was used in the preparation of YFHXG serum. Tissue block anchorage was employed in the primary culture of rat PASMCs. The PASMCs were randomly divided into normoxia group, hypoxia group, and hypoxia+YFHXG group (0.66, 3.30 and 16.50 g/kg doses of YFHXG-treated serum groups, exposed to hypoxic condition). PASMCs in normoxia and hypoxia group were cultured with saline serum, hypoxia+YFHXG groups were cultured with different concentrations of YFHXG serum. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle was analyzed using flow cytometry. In addition, hypoxia inducible factor-1-alpha (HIF-1α) protein expression was evaluated by immunocytochemistry analysis, the concentration of intracellular reactive oxygen species (ROS) and Ca(2+) were determined by laser scanning confocal microscopy (LSCM).

RESULTSMTT assay and flow cytometry showed that hypoxia could directly activate the proliferation of PASMCs, while YFHXG dose-dependently inhibited hypoxia-induced proliferation of rat PASMCs. Immunocytochemistry showed that hypoxia enhanced HIF-1α protein expression, and LSCM showed that hypoxia significantly increased intracellular ROS and Ca(2+), while YFHXG decreased the expression of HIF- 1α and attenuated the hypoxia-induced increase in intracellular concentration of ROS and Ca(2+).

CONCLUSIONSYFHXG could inhibit hypoxia-induced proliferation of rat PASMCs, which may decrease pulmonary arterial pressure and vascular remodeling. The anti-hypoxia effect of YFHXG may be explained by its regulation of HIF-1α expression and of the levels of intracellular ROS and Ca(2+).

Animals ; Calcium ; metabolism ; Cell Cycle ; drug effects ; Cell Hypoxia ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Intracellular Space ; drug effects ; metabolism ; Male ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

PURPOSE: Pulmonary Kv channels are thought to play a crucial role in the regulation of cell proliferation and apoptosis. Previous studies have shown that fluoxetine upregulated the expression of Kv1.5 and prevented pulmonary arterial hypertension in monocrotaline-induced or hypoxia-induced rats and mice. The current study was designed to test how fluoxetine regulates Kv1.5 channels, subsequently promoting apoptosis in human PASMCs cultured in vitro. MATERIALS AND METHODS: Human PASMCs were incubated with low-serum DMEM, ET-1, and fluoxetine with and without ET-1 separately for 72 h. Then the proliferation, apoptosis, and expression of TRPC1 and Kv1.5 were detected. RESULTS: In the ET-1 induced group, the upregulation of TRPC1 and down regulation of Kv1.5 enhanced proliferation and anti-apoptosis, which was reversed when treated with fluoxetine. The decreased expression of TRPC1 increased the expression of Kv1.5, subsequently inhibiting proliferation while promoting apoptosis. CONCLUSION: The results from the present study suggested that fluoxetine protects against big endothelin-1 induced anti-apoptosis and rescues Kv1.5 channels in human pulmonary arterial smooth muscle cells, potentially by decreasing intracellular concentrations of Ca2+.
Apoptosis/drug effects/genetics ; Blotting, Western ; Cell Proliferation/drug effects ; Cells, Cultured ; Endothelin-1/*pharmacology ; Flow Cytometry ; Fluoxetine/*pharmacology ; Humans ; Kv1.5 Potassium Channel/genetics/*metabolism ; Muscle, Smooth, Vascular/*cytology/drug effects ; Pulmonary Artery/*cytology ; Reverse Transcriptase Polymerase Chain Reaction