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.

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.

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.

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.

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.

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.

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.

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.

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

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.

Study of the mechanisms regulating human umbilical artery contractility

We studied the involvement of different types of Ca2+ channels, cyclic nucleotides and different kinases in the regulation of human umbilical artery (HUA) contractility. The elucidation of the precise mechanisms regulating the contractility of this artery could be very important to reveal potential therapeutic targets to treat HUA disorders such as preeclampsia. The relevancy of different types of Ca2+ channels on the regulation of HUA tonus was analyzed. Among the different Ca2+ channel inhibitors used, only the L-type calcium channels (LTCC) inhibition induced relaxation of HUA in Ca2+ containing medium. The inhibition of T-type calcium channels (TTCC) or TRP channels did not significantly affect HUA contractility. In presence of Ca2+, the intracellular increase of a cyclic nucleotide (cAMP or cGMP) induces relaxation of HUA, which was almost complete in histamine-con- tracted HUA, and lower effect was observed in arteries contracted by KCl and serotonin (5-HT). Inhibition of PKA and PKG weakly reduced the relaxations induced by the increase of cAMP and cGMP respectively, suggesting that the relaxation induced by these nucleotides is not totally mediated by the activation of their respective kinases and that other mechanisms are involved. In calcium containing solution, PP2A inhibition produces relaxation of contracted HUA. In KCl contracted arteries, the OA and nifedipine relaxant effects are similar and not additive, suggesting that PP2A could activate LTCC. Besides, the increase of cyclic nucleotides significantly increased the OA effect, suggesting that the effect of PP2A inhibition is independent of the cyclic nucleotide pathways. The contractions induced by KCl, histamine and 5-HT in presence of Ca2+ were not significantly affected by ROCK, ERK1/2 or p38MAPK inhibitors. In absence of extracellular Ca2+, histamine and 5-HT elicited contractions of HUA characterized by two components, a rapid phasic contractile component followed by a decrease of the contraction until a tonic component. However, KCl elicited sustained contractions of HUA in absence of extracellular Ca2+. As in presence of calcium, the ERK1/2 and p38MAPK inhibitors did not influence the contractions induced by KCl, histamine or 5-HT in absence of extracellular Ca2+. However, in these conditions, ROCK inhibition significantly relaxed the contractions induced by KCl and reduced the phasic and tonic components of the contraction elicited either by histamine or 5-HT. Our results show that calcium-dependent contractions of HUA depend on Ca2+ entry by LTCC, and these chan- nels seems to be positive regulated by PP2A. Cyclic nucleotides mediate HUA vasodilatation but their dependent kinases are not the unique responsible of this effect. HUA is able to contract independently of Ca2+ influx by activating the ROCK pathway and/or due to intracellular Ca2+ release.

Immuohistochemical study on smooth muscle cell proliferation, pheno-typic modulation, and extracellular matrix accumulation in venous arterial grafts in rabbits

Objective: To study the kinetics and distribution of smooth muscle cell (SMC) proliferation, phe-notypic modulation, and various extracellular matrix (ECM) components accumulation during vein graft remodeling. Methods: Normal vein and vein graft in carotid arteries were examined on d 4, d 7, d 14, d 60 and d180 after bypass grafting with immunohistochemical markers of cellular proliferation (proliferating cell nuclear antigen, PCNA), cytoskeletal protein production (a-actin SMC), myosin heavy chain (MHO iso-forms, ECM proteins, and histochemistry (hematoxylin eosin and Elastica-van Gieson stain). Results: Normal veins demonstrated an extremely low level of cellular proliferation and expressed as adult phenotype SM-Cs in media. After bypass grafting, medial SMCs in the graft appeared to be damaged and began to proliferate on d 4, and subsequently migrated and formed the neointima on d 7. Thereafter, the neointima thickened throughout the 180-day period of the experiment, although the neointimal SMC proliferation decreased after d 14. Meanwhile SMCs underwent a distinct phenotypic change from normal adult type to embryonic type. On d 60, embryonic phenotype SMCs began to return to the adult phenotype, but remain to be present in the neointima for as long as 180 d. ECM components including type I collagen, heparin sulfate proteoglucan (HSPG), and dermatan sulfate proteoglcan (decorin) were detected within the neointima on d 7. Thereafter, the accumulation of ECM increased progressively with time. On d 180, a large amount of ECM components were found in the neointima. HSPG mainly accumulated in the superficial and cellular region of the neointima , decorin, on other hand, located in hypocellular area deep in neointima. Type I collagen scatted in both regions. The elastic fibers became rich and arranged continuously in the neointima. Conclusion: The neointima of vein graft was initially formed by proliferation of the embryonic-type SMCs and then thickened infinitely due to ECM accumulation. Prolonged existence of the embryonic-type SMCs in the neointima may contribute to ECM accumulation and increase in the neointima thickness infinitely, which may predispose accelerated stenosis in the vein graft.

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.

New categorization of human vascular endothelial cells by pro-vs anti-proliferative phenotypes

AIM: To integrally understand the effects of human vascular endothelial cells(VECs) on the proliferation of vascular smooth muscle cells(VSMCs).METHODS: Various kinds of human VECs of different origins were co-cultured with human aortic smooth muscle cells, a representative of human VSMCs. To exclude the irrelevant effects due to growth competition between VECs and VSMCs, the proliferation of VECs had previously been arrested via a low-dose gamma rayirradiation. To discriminately analyze the proliferation of VSMCs from that of VECs, the former cells were labeled with red fluorescent dye while the latter cells were labeled with green fluorescent dye before performing coculture experiments. After 4 d, total cells were harvested and subjected to flow cytometric analyses. Decrements in red fluorescence intensities due to proliferationmediated dilutions were measured and mathematically processed using a specific software to quantitatively evaluate the proliferation of VSMCs. The findings obtained from the flow cytometry-based analyses were further validated by microscopic observations. RESULTS: Commercially available primary cultured human VECs exclusively promoted VSMC proliferation regardless of their tissue origins and we termed these pro-proliferative VECs as "typeⅠ". By contrast, VECs freshly generated from human bone marrow-derived endothelial progenitors cells or human pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells suppressed VSMC proliferation and we termed these anti-proliferative VECs as "typeⅡ". Repetitive subcultures as well as oxidative stress induced "type Ⅱ VECs to typeⅠ" conversion along with an induction of Regulator of G-protein signaling 5(RGS5)Compatibly, anti-oxidant treatments suppressed both the subculture-dependent "typeⅡ to typeⅠ" conversion and an induction of RGS5 gene. Immunostaining studies of clinical specimens indicated that RGS5 protein expressions in endothelial layers were low in norma arteries but they were up-regulated in pathologica arteries including hypertension, atherosclerosis and autoimmune vasculitis in a dose-dependent manner Overexpression and knockdown of RGS5 caused that"typeⅡ to typeⅠ" and "typeⅠ to type Ⅱ" phenotype conversions of VECs, respectively. CONCLUSION: Human VECs are categorized into two types: pro-proliferative RGS5^(high) VECs(typeⅠ) and antiproliferative RGS5 ^(low) VECs(typeⅡ).

Iododeoxyuridine uptake in proliferating smooth musc le cells:an in vitro model to assess drug effects on intimal hyperplasia

Purpose To assess the maximum uptake of Iododeo xyur idine (IUdR) by proliferating smooth muscle cells in vitro to determine the opti mal concentration to be administrated in an in vivo experiment. The long-term g oal is to utilize radioactive IUdR to inhibit smooth muscle cell proliferation a nd restenosis of arteries after balloon angioplasty in vivo. Methods Porcine smooth muscle cells (SMCs) were cultured in 5% FBS medium and stim ulated to proliferate by the addition of medium containing 10% FBS and insulin. IUdR was added at 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, respectively, in prolif erating SMCs with control for 1, 3, 5, 7 day incubation. Fluorescence Activated Cell Scanning (FACS) was performed after the SMCs were harvested and double-sta ined with FITC-conjugated anti-IUdR antibody (B44) and propidium iodide (PI). The ratio of IUdR-labeled cells to total cell population for each IUdR concentr ation and duration was determined by FACS. All data were repeated three times at each time point. The doubling times, growth curve and cell density of the proli ferating SMCs were investigated using Beckman Coulter Particle Counter and digit al microscopy. Results The percentage of proliferating SMCs uptaking IUdR incr eased from 1 to 5 days incubation with all concentrations of IUdR; In day 5, the uptake rate reached the peak value, then decreased by 7 days. IUdR uptake on d ay 5 was higher with concentrations of 10 μM and 20 μM. The doubling times of the SMCs were prolonged with IUdR concentration increasing, while the proliferat ing cell number and density compared with control decreased obviously by day 5 ( P<0.05).Conclusion The peak time to uptake IUdR was 5 days and optimal concentration of IUdR was between10 μM to 20 μM for proliferating SMCs to upta ke in vitro. IUdR itself could inhibit the SMCs’ proliferation and the inhibito ry effect was related to the concentration.[