Involvement of TRPC1 and Cyclin D1 in Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced by Cigarette Smoke Extract

Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.

Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

In order to investigate the effect of nuclear factor kappa B (NF κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF κB. The NF κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBα protein expression was measured by Western blot. RT PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups ( P <0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups ( P <0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration dependent manner in hypoxia. In conclusion, NF κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF κB activation can decrease the VEGF mRNA expression. It is suggested that the activation of NF κB is involved in the VEGF mRNA expression of HPASMCs under hypoxia.

Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs) We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense FAK respectively Expression of FAK, Jun NH2 terminal kinase (JNK), cyclin dependent kinase 2 (CDK 2) and caspase 3 proteins were detected by immunoprecipitation and Western blots Cell cycle and cell apoptosis were analysed by flow cytometry In addition, cytoplasmic FAK expression was detected by immunocytochemical staining Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense FAK ODNs group and increased in sense FAK ODNs group significantly Caspase 3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs When compared with mismatch sense ODNs group, the proportion of cells at G 1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly In contrast, compared with mismatch sense ODNs group, the proportion of cells at G 1 phase was increased significantly in antisense FAK ODNs group The level of cell apoptosis in antisense FAK group was higher than in the mismatch sense group and the latter was higher than sense FAK group In addition, the sense FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense FAK ODNs group was weakly stained Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase 3 inhibits HPASMCs apoptosis

NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1αexpression

Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.

Fasudil inhibits platelet-derived growth factor-induced human pulmonary artery smooth muscle cell proliferation by up-regulation of p27^kipl via the ERK signal pathway

Background RhoA/ Rho kinase （ROCK） pathway is involved in pulmonary arterial hypertension （PAH） and pulmonary artery smooth muscle cell （PASMC） proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor （PDGF） induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase （ERK）,p27KiP1.Methods Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen （PCNA） and p27Kip1 were measured by immunoblotting.Results By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G0/G1 phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27Kip1 expression in human PASMCs, which decreased after PDGF stimulation.Conclusion RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27Kip1, which may be associated with inhibition of ERK activity.

Effect of mitochondrial K_(ATP) channel on voltage gated K^+ channel in 24 hour-hypoxic human pulmonary artery smooth muscle cells

Background Hypoxic pulmonary hypertension (HPH) is initiated by inhibition of O 2 sensitive, voltage gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs) The mechanism of hypoxic pulmonary hypertension has not yet been fully elucidated The mitochondrial ATP sensitive K + channel (MitoK ATP ) is extremely sensitive to hypoxia, and is a decisive factor in the control of mitochondrial membrane potential (ΔΨ m) This study investigated the changes of cell membrane potential and Kv channel in cultured human pulmonary artery smooth muscle cell (hPASMC) exposed to 24 hour hypoxia, and explored the role of MitoK ATP and ΔΨ m in this condition Methods Fresh human lung tissues were obtained from the patients undergoing a chest operation hPASMCs were isolated, cultured, and divided into 6 groups: ① control group, cultured under normoxia; ② diazoxide group, cultured in normoxia with diazoxide, an opener of MitoK ATP ; ③ 5 HD group, cultured in normoxia with sodium 5 hydroxydecanoate (5 HD), an antagonist of MitoK ATP ; ④ 24 hour hypoxia group; ⑤ 24 hour hypoxia + diazoxide group; and ⑥ 24 hour hypoxia + 5HD group Whole cell patch clamp technique was used to trace the cell membrane K + currents The expressions of cell membrane Kv1 5 mRNA and protein were determined by RT PCR and Western blot technique, respectively The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R 123) technique Results After exposure to diazoxide for 24 hours, the intensity of R 123 fluorescence in normoxic hPASMCs was significantly increased compared with control group ( P <0 05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5 HD for 24 hours Twenty four hour hypoxia or 24 hour hypoxia + diazoxide could markedly increase the intensity of R 123 fluorescence in hPASMC and the changes were more significant in 24 hour hypoxia +diazoxide group than in 24 hour hypoxia group ( P <0 05) although 5 HD could partly weaken the effect of 24 hour hypoxia on the intensity of R 123 fluorescence After exposure to diazoxide for 24 hours, the cell membrane K + currents and the expression of cell membrane Kv1 5 mRNA and protein in normoxic hPASMCs were significantly decreased compared with control group ( P <0 05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5 HD for 24 hours Also, 24 hour hypoxia or 24 hour hypoxia + diazoxide decreased the cell membrane K + currents and the expression of Kv1 5 mRNA and protein ( P <0 05) but the changes were more significant in 24 hour hypoxia + diazoxide group than in 24 hour hypoxia group ( P <0 05) Again, 5 HD could partly weaken the inhibitory effect of 24 hour hypoxia on the cell membrane K + currents and the expression of Kv1 5 mRNA or protein ( P <0 05) Conclusions The opening of MitoK ATP followed by a depolarization of ΔΨ m in hypoxia might contribute to the alterations in the expression of cell membrane Kv1 5 mRNA and protein leading to change in the cell membrane potential of hypoxic hPASMCs This might be a mechanism of the development of hypoxic pulmonary hypertension

Inhibitory Effect of PPARδAgonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway

Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.

Relationship of Intracellular Free Ca^(2+) Concentration and Calcium-activated Chloride Channels of Pulmonary Artery Smooth Muscle Cells in Rats under Hypoxic Conditions

To investigate the relationship between intracellular free Ca^2＋ concentration （[Ca^2＋ ]i ） and calcium-activated chloride （Clca） channels of pulmonary artery smooth muscle cells （PASMCs） in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca^2＋ indicator Fura-2/AM was used to observe [Ca^2＋ ]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay. The Clca channel blockers niflumic acid （NFA） and indaryloxyacetic acid （IAA-94） exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca^2＋ ]i was increased. Under normoxic condition, [Ca^2＋ If was （123.634-18.98） nmol/ L, and in hypoxic condition, [Ca^2＋]i wag （281. 754-16.48） nmol/L （P〈0. 01）. Under normoxic condition, [Ca^2＋ ]i showed no significant change and no effect on Clca channels was observed （P〉 0. 05）. Chronic hypoxia increased [Ca^2＋ ]i which opened Clca channels. The NFA and IAA-94 blocked the channels and decreased [Ca^2＋ ]i from （281.75± 16.48） nmot/L to （117.66 ±15.36） nmol/L （P〈0.01）. MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency （A value） from 0. 459±0. 058 to 0. 224±0. 025 （P〈0. 01）. Hypoxia increased [Ca^2＋ ]i which opened Cl~ channels and had a positive-feedback in [Ca^2＋ ]i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Clca channel may play a part in the regulation of proliferation of PASMCs.

The Effect of Erigeron Breviscapus on Proliferation of Pulmonary Artery Smooth Muscle Cells in Hypoxic Porcines

In order to study the effect of Erigeron Breviscapus (EB) on proliferation of pulmonary artery smooth muscle cells (PASMC) in hypoxic porcines, immunohistochemical and MTT methods were employed to measure the proliferation of PASMC. It was found that the proliferation of PASMC in porcines was obvious, and the expression of proliferating cell nuclear antigen (PCNA) was significantly high within 48 h after exposure to hypoxia. The EB could inhibit the proliferation and the expression of PCNA in PASMC under hypoxia, but it had no effect on the proliferation and expression of PCNA in PASMC under normal condition. The EB could inhibit the proliferation and the expression of PCNA in PASMC induced by phorbol 12-myristate 13-acetate (PMA), an agonist of PKC in normal and hypoxic conditions. It was concluded that the hypoxia could enhance the proliferation and expression of PCNA in PASMC. The EB can inhibit the proliferation and expression of PCNA in PASMC under hypoxia through PKC-signal way. The EB may be used in treating the pulmonary hypertension by inhibiting the proliferation of PASMC and the pulmonary vascular remodeling.

Hypoxia Down-regulates Secretion of MMP-2, MMP-9 in Porcine Pulmonary Artery Endothelial and Smooth Muscle Cells and the Role of HIF-1

Summary： 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 he mitigated by the transfection of EPO3 ＇-enhancer and that H1F-1 pathway might contribute to hypoxia-induced down regulation of MMP-2 and MMP-9.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective：To investigate the effects of calcium-activated chloride （ClCa） channels on proliferation of pulmonary artery smooth muscle cells（PASMCs） in rats under chronic hypoxic condition. Methods：The cultured PASMCs were placed under normoxic and chronic hypoxic conditions：The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca^2＋ concentration （[Ca^2＋]i） in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results：The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope： In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi＇s bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S ＋G2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from （28.6±1.0）% to （16.0±1.6）% and the number of G0G1 PASMCs significantly increased from （71.4± 1.9）% to （83.9 ± 1.6）% （P〈 0.01）. In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from （81 ± 6）% to （27 ± 7）%（P 〈 0.01）. The expression of c-fos and c-jun were significantly increased in＇chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ±0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively （P〈 0.01）; Under chronic hypoxic conditions, [Ca^2＋]i was increased; The NFA and IAA-94 decreased it from （281.8±16,5）nmol/L to （117.7 ± 15.4）nmol/L（P 〈 0.01）. Conclusion：Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

EFFECT OF HYPOXIA ON DNA SYNTHESIS AND C－MYC GENE EXPRESSION OF PULMONARY ARTERY SMOOTH MUSCLE CELLS

The neonate is particularly susceptible to the development of hypoxic pulmonary hypertension. The present study was undertaken to observe the effect of hypoxia on DNA synthesis and c-myc gene expression between newborn calf and adult bovine PASMC in vilro. DNA synthesis measured by 3H- TdR incorporation was increased after hypoxic challenge for 24h. Hypoxia enhanced the increment in 3H-TdR incorporation induced by EGF. Northern blot analysis revealed that PASMC cultured in both normoxia and hypoxia expressed c- myc gene transcript of 2. 2Kb ,but there is a higher 2. 2Kb mRNA expression in hypoxic PASMC than that in normoxia. We speculate that newborn calf PASMC exhibited potential response to hypoxia than adult,which was augmented by EGF. Enhanced c-myc gene expression may lead to a great understanding of the mechanism of PASMC growth in the development of pulmonary hypertension.

Effects of NHE-1 ribozyme gene transfection on apoptosis of rat pulmonary artery smooth muscle cells in vitro

Objective : To investigate the effects of the transfection of NHE-1 ribozyme gene on the apoptosis of pulmonary artery smooth muscle cells (PASMC) in vitro. Methods: After NHE-1 ribozyme gene was designed, synthesized and then cloned into plasmid pLXSN, the recombined plasmid was tansfected into cultured rat PASMC. Expression of NHE-1 mRNA was detected with semi-quantitative RT-PCR. Intracellular pH (pHi) was measured by using fluorescence dye BCECF-AM. Cell cycle was measured with aid of flow cy-tometric DNA analysis. Cell apoptosis was observed with electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNED respectively. Results: The NHE-1 mRNA expression level and pHi value were significantly lower in PASMCs transfected with NHE-1 ribozyme gene than those transfected with pLXSN or without transfection. Meanwhile, the apoptosis rate of cells transfected with NHE-1 ribozyme gene was increased significantly. Morphology of cell apoptosis was observed in the cells transfected with NHE-1 ribozyme gene under an electron microscope. Conclusion: The transfection of NHE-1 ribozyme gene induces the apoptosis of PASMCs by inhibiting NHE-1 expression and intracellular acidification.

Iptakalim, a novel ATP-sensitive potassium channel opener, inhibits pulmonary arterial smooth muscle cell proliferation by downregulation of PKC-α

Iptakalim is a new ATP-sensitive potassium （KATp） channel opener, and it inhibits the proliferation of pulmonary arterial smooth muscle cells （PASMCs） and pulmonary vascular remodeling. However, the underlying mechanism remains unclear. In the present study, we found that iptakalim significantly decreased pulmonary artery pressure, inhibited pulmonary ariery remodeling and PKC-α overexpression in chronic hypoxia in a rat pulmonary hypertension model. Iptakalim reduced hypoxia-induced expression of PKC-α, and abolished the effect of hypoxia on PASMC proliferation significantly in a dose-dependent manner in vitro. Moreover, these effects were abol- ished by glibenclamide, a selective KArp channel antagonist. These results indicate that iptakalim inhibits PASMC proliferation and pulmonary vascular remodeling induced by hypoxia through downregulating the expression of PKC-α. Iptakalim can serve as a novel promising treatment for hypoxic pulmonary hypertension.

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering

BACKGROUND The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations.Mesenchymal stromal cells(MSCs) derived from the Wharton's Jelly(WJ) tissue can be used as a source for obtaining vascular smooth muscle cells(VSMCs),while the human umbilical arteries(h UAs) can serve as a scaffold for blood vessel engineering.AIM To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate.METHODS WJ-MSCs were isolated and expanded until passage(P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid,followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9,NANOG homeobox, OCT4 and GAPDH, was performed. In addition,immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated h UAs.RESULTS WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into"osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression(> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, h UAs were isolated and decellularized. Based on histological analysis, decellularized h UAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized h UAs with VSMCs was performed for 3 wk. Decellularized h UAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and s GAG contents in repopulated h UAs with VSMCs. Specifically, total hydroxyproline and s GAG content after the 1 st, 2 nd and 3 rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 μg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1μg s GAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups(P<0.05).CONCLUSION VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, h UAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.

Hypoxia promotes cell proliferation by modulating E2F1 in chicken pulmonary arterial smooth muscle cells

In this study,we sought to investigate the expression of the transcription factor E2F1 in chicken pulmonary arterial smooth muscle cells upon hypoxia exposure,as well as the role that E2F1 played in the regulation of cell proliferation.Isolated chicken pulmonary arterial smooth muscle cells were subjected to hypoxia or normoxia for indicated time points.Cell viability,DNA synthesis,cell cycle profile,and expression of E2F1 were analyzed.The results showed that hypoxia promoted cell proliferation and DNA synthesis which was accompanied by an increased S phase entry and upregulation of E2F1 at mRNA and protein levels.Using siRNA technology,we demonstrated that gene inactivation of endogenous E2F1 abolished hypoxia-induced cell proliferation,DNA synthesis,and S phase entry compared with negative siRNA transfected cells.These results suggest that hypoxia-induced proliferation is mediated by inducing E2F1 in chicken pulmonary arterial smooth muscle cells.

Role of Na^+/H^+ antiporter in pulmonary artery smooth muscle of hypoxic pulmonary hypertension in the rat

Objective: To investigate the role of Na+ /H+ antiporter in the hypoxic pumonary hypertension ofrats. Methods: Thirty Wistar rats were randomly divided into 3 groups with 10 in each group: controlgroup, 3--week hypoxia group and 8--week hypoxia group. After the isolation of pulmonary artery smoothmuscles, pHi was determined by fluorescence measurement of the pH--sensitive dye BCECF and theexpression of NHE--1 mRNA was detected with reverse transcription--polymerase chain reaction. Results: ThepHi and expression of NHE-1 mRNA of pulmonary artery smooth muscle cell in the hypoxia groups weresignificantly increased than those in the normal group (P < 0. 01 ). There was no remarkable differencebetween the hypoxia groups. Conclusion: With the function of regulation pHi., NHE--1 may play an importantrole in the pulmonary vascular remodeling of pulmonary hypertension. The result provides a new therapeuticmethod with NHE--1 inhibitors and/or gene therapy for the hypoxic pulmonary hypertension.

Oxidative modification of high density lipoprotein induced by cultured human arterial smooth muscle cells

Objective: To observe the oxidative modification of high density lipoprotein (HDL) induced by cultured human arterial smooth muscle cells (SMCs). Methods: HDL cocultured with SMCs at 37℃ in 48 h was subjected, and native HDL (N-HDL) served as control. Oxidative modification of HDL was identified by using agarose gel electrophoresis. Absorbances of conjugated diene (CD) and lipid hydroperoxide (LOOH) were measured with ultraviolet spectrophotometry at 234 and 560 nm respectively, and fluorescence intensity of thiobarbuturic acid reaction substance (TBARS) with fluorescence spectrophotometry at 550 nm emission wavelength with excitation at 515 nm. Results: In comparison with N-HDL, the electrophoretic mobility of SMCs-cocultured HDL was increased, and the contents of CD, LOOH and TBARS HDL were very significantly higher than those of the control HDL (P<0.01). Conclusion: Oxidative modification of HDL can be induced by human arterial SMCs.

The inhibitory effect of endogenous opioid peptide on the proliferation of pulmonary arterial smooth muscle cells

To investigate the effects of endogenous opioid peptides L-enkephalin (L-Enk) and morphine on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) and analyze their mechanism. Methods: Rabbit PASMCs cultured invitro, MTT method and 3 H-TdR incoporation were Used. Results: 1×10-3 -1×10-4 mol/L L-Enk markedly inhibited the proliferation and the DNA synthesis of PALMCs, which could be reversed by naloxone, an opioid receptor antagonist, while orphineseemed to have no obvious effects on the proliferation and the DNA synthesis of PASMCs. Conclusion: Endogenous opioid peptidecan inhibit the proliferation and DNA synthesis of PASMCs, which is mainly mediated through opioid δ receptor and not opioid μreceptor.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells:Role of cystathionineγ-lyase/hydrogen sulfide pathway

BACKGROUND Ferroptosis can induce low retention and engraftment after mesenchymal stem cell(MSC)delivery,which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension(PAH)therapy.Interestingly,the cystathionineγ-lyase(CSE)/hydrogen sulfide(H_(2)S)pathway may contribute to mediating ferroptosis.However,the influence of the CSE/H_(2)S pathway on ferroptosis in human umbilical cord MSCs(HUCMSCs)remains unclear.AIM To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H_(2)S pathway-mediated ferroptosis,and to investigate the functions of the CSE/H_(2)S pathway in ferroptosis in HUCMSCs and the underlying mechanisms.METHODS Erastin and ferrostatin-1(Fer-1)were used to induce and inhibit ferroptosis,respectively.HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE.A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions,and pulmonary pressure and vascular remodelling were measured.The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging.Cell viability,iron accumulation,reactive oxygen species production,cystine uptake,and lipid peroxidation in HUCMSCs were tested.Ferroptosis-related proteins and S-sulfhydrated Kelchlike ECH-associating protein 1(Keap1)were detected by western blot analysis.RESULTS In vivo,CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxiainduced PAH.In vitro,CSE overexpression improved H_(2)S production and ferroptosis-related indexes,such as cell viability,iron level,reactive oxygen species production,cystine uptake,lipid peroxidation,mitochondrial membrane density,and ferroptosis-related protein expression,in erastin-treated HUCMSCs.In contrast,in vivo,CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice.In vitro,CSE inhibition decreased H_(2)S levels and restored ferroptosis in Fer-1-treated HUCMSCs.Interestingly,upregulation of the CSE/H_(2)S pathway induced Keap1 S-sulfhydration,which contributed to the inhibition of ferroptosis.CONCLUSION Regulation of the CSE/H_(2)S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH.Moreover,the protective effect of the CSE/H_(2)S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling.The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.