Effects of She Xiang Bao Xin Wan (SXBXW) on the proliferation of primary cultured human umbilical artery vascular smooth muscle cells induced by endothelin-1

AIM: To observe the effects of She Xiang Bao Xin Wan (SXBXW) of Chinese patent medicine on the proliferation of primary cultured vascular smooth muscle cells (VSMCs) from human umbilical artery and stimulated by endothelin-1 (ET-1) in vitro. METHODS: The proliferation cell models of primary cultured VSMCs were established by ET-1 stimulation. Six groups in the experiment were divided into control group; ET-1 group; ET-1+SXBXW 0.25 g/L; ET-1+SXBXW 0.5 g/L; ET-1+SXBXW 1.0 g/L and ET-1+SXBXW 2.0 g/L groups, respectively. The proliferation induced by ET-1 and the suppression mediated by SXBXW on VSMCs were measured by MTT method. The inhibitory rate and the cytotoxicity of SXBXW were detected by lactate dehydrogenase colorimetry and trypan blue exclusion tests. The effect of ET-1 and SXBXW on the cell proliferation cycle was analyzed by flow cytometry. RESULTS: Compared to control group, ET-1 significantly enhanced the proliferation of VSMCs (P<0.01). However, a certain dose of SXBXW inhibited effectively the proliferation of VSMCs induced by ET-1 in a dose-dependent manner (P<0.01). Meanwhile, SXBXW showed no influenced on both the number of living cells and the release of lactate dehydrogenase, although it inhibited the proliferation of VSMCs, indicating that SXBXW was no cytotoxicitic effect on VSMCs. ET-1 enhanced the proliferation of VSMCs by means of promoting the transition of the cell cycle from G1 phase to S phase. However SXBXW significantly inhibited the proliferation mediated by ET-1. CONCLUSION: SXBXW plays the role in suppressing VSMCs proliferation induced by ET-1. The mechanism may be involved in blocking the cell cycle from G1 phase into S phase.     

NKT cells inhibit Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by down-regulating PDGFRβ

AIM To investigate the role of natural killer T (NKT) cells in the phenotypic transformation of vascular smooth muscle cells induced by hypertension in mice. METHODS The hypertension model in wild-type and CD1d gene knockout mice was established by continuous infusion of angiotensin II (Ang II) at a dose of 490 ng·kg^-1·min^-1 for 14 d, and the NKT cell specific agonist α-galactosylceramide (α-GC) was given at a dose of 100 μg/kg in wild-type mice. The blood pressure of the mice was monitored by non-invasive tail cuff method. The thickness of aortic wall was measured by HE staining, and the degree of aortic fibrosis was observed by Masson staining. The expression levels of α-smooth muscle actin (α-SMA), osteopontin (OPN) and platelet-derived growth factor receptor β (PDGFRβ) in aortic tissue were detected by Western blot. RESULTS Compared with control group, the blood pressure, the aortic wall thickness, the degree of aortic fibrosis, the secretory marker protein OPN expression and the PDGFRβ expression were all increased significantly after infusion of Ang II for 14 d (P<0.05), while the expression of contractile marker protein α-SMA was decreased (P<0.05). The above changes were aggravated after CD1d gene knockout (P<0.05), but were attenuated after administration of α-GC (P<0.05). CONCLUSION NKT cells reduces Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by reducing the expression of PDGFRβ, increasing the expression of contractile protein and decreasing the expression of secretory protein.     

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.     

Model construction of rat coronary artery smooth muscle cell endoplasmic reticulum stress induced by thapsigargin

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress (ERS) model in CASMCs of SD rats. METHODS: CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs, including α-SMA and SM-MHC, were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP, the marker molecules of ERS, were determined by Western blot. RESULTS: The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical "hill and valley" growth pattern of CASMCs at 9~10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG (1 and 2 μmol/L) treatment groups was increased compared with control group. Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION: CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.     

Mechanism of quercetin improving rat coronary artery myogenic response under high glucose

AIM: To investigate the mechanism of quercetin improving rat coronary artery myogenic response under high glucose (HG) by measuring muscle tension of coronary arterial ring and recording voltage-gated K⁺ channel (Kv) current of coronary artery smooth muscle cells by whole cell patch clamp. METHODS: The coronary rings from the normal SD rats were acutely isolated, and then divided into 6 groups: (1) control group; (2) HG group; (3) HG+low dose (3 μmol/L) of quercetin group; (4) HG+moderate dose (10 μmol/L) of quercetin group; (5) HG+high dose (30 μmol/L) of quercetin group; (6) HG+C6303 (PKC inhibitor)+high dose of quercetin group. Determinations of coronary artery response to vasoconstrictor (60 mmol/L KCl or 0.1 mmol/L U46619) or vasodilator (ACh at 10^-9~10^-5 mol/L) were performed, and the percentage of coronary ring tension was calculated using the contraction as 100% caused by 60 mmol/L KCl. The rat coronary artery smooth muscle cells were acutely isolated for recording the Kv current using whole cell patch clamp. RESULTS: Compared with control group, the contraction amplitudes to 60 mmol/L KCl or 0.1 mmol/L U46619 were significantly increased under HG incubation. Quercetin intervention concentration-dependently reduced the coronary artery contraction amplitude. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. Compared with control group, the diastolic amplitude to ACh decreased significantly in HG group, and quercetin intervention concentration-dependently increased the coronary artery diastolic amplitude. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. Compared with control group, HG incubation inhibited Kv current of coronary artery vascular smooth muscle cells significantly, and quercetin intervention attenuated the inhibitory effect of HG on Kv current intensity. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. CONCLUSION: Quercetin has a protective effect on myogenic response of coronary artery under HG and the effects is related to the increase in Kv current and the activation of PKC in vascular smooth muscle cells.     

Changes of Ca2＋ activated potassium channels and cellular-proliferation in autogenous vein grafts

AIM: To investigate changes of Ca^2＋ activated potassium channels (K_Ca) in autogenous vein grafts. METHODS: The contraction of venous ring was measured by means of perfusion in vitro. The intimal proliferation and proliferation of cultured smooth muscle cells (vascular smooth muscle cells, VSMCs) were observed by the means of computerised image analysis and MTT method, respectively. Furthermore, whole cell mode of patch clamp was used to record K_Ca of VSMCs isolated from autogenous vein grafts. RESULTS: 1 week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P＜0.05, n=8 vs control), and they were more enhanced 4 weeks after vein transplantation (P＜0.01, n=8 vs control). TEA (blocker of Ca^2＋ activated potassium channels) increased MTT A₄₉₀ value of VSMCs from femoral vein in a dose-dependent manner (P＜0.05, n=8). K_Ca current density was significantly attenuated in VSMCs from autogenous vein grafts 1-4 weeks after transplantation (P＜0.05, n=5). CONCLUSION: K_Ca was inhibited in autogenous vein graft, which accounted for vasospasm and intimal proliferation.     

Effects of extract of ginkgo biloba on human tubular epithelial-mesenchymal transition induced by transforming growth factor-β1

AIM: to investigate the effects of extract of ginkgo biloba (EGB) on human tubular epithelial-mesenchymal transition induced by transforming growth factor-β₁.METHODS: HK2 cells were induced to epithelial-mesenchymal transition by transforming growth factor-β₁ (TGF-β₁, 10 μg/L). EGB was added into the medium of HK2 cells 2 h before TGF-β₁ was added. The expressions of E-cadherin, α-smooth muscle actin (α-SMA), NADPH oxidase p67phox and superoxide dismutase (SOD) were determined by Western blotting. Malondialdehyde (MDA) in the mediums of HK2 cells was detected. RESULTS: EGB significantly attenuated the downregulation of E-cadherin, the upregulation of α-SMA and p67phox, the downregulation of SOD and the upregulation of MDA in HK2 cells induced by TGF-β₁.CONCLUSION: EGB significantly attenuates human tubular epithelial-mesenchymal transition induced by TGF-β₁, and its underlying mechanism is that EGB attenuates the upregulation of p67phox and the downregulation of SOD induced by TGF-β₁.     

Effects of marine fungal metabolites 1386A from the South China Sea on proliferation,apoptosis and mitochondrial membrane potential in gastric cancer cell line MCG-803

AIM: To investigate the effects of marine fungal metabolites 1386A from the South China Sea(1386A) on gastric cancer cell line MCG-803.METHODS: The effects of 1386A on cell viability and cell growth were detected by MTT method and colony-forming assay, respectively. The changes of cell cycle, cell apoptosis and mitochondrial membrane potential were determined by flow cytometry.RESULTS: The IC₅₀ of cell viability on MCG-803 cells after treated with 1386A for 24 h, 48 h and 72 h was 29.70 μmol/L, 14.07 μmol/L or 13.47 μmol/L, respectively. The IC₅₀ of the cell growth on MCG-803 cells after treated with 1386A for 48 h was 3.27 μmol/L. After treated with 1386A for 48 h, the MCG-803 cells in S stage was increased from 25.6% to 56.8%. After treated with 1386A for 24 h, the early apoptosis of MCG-803 cells was increased from 3.34% to 8.39%, and the mitochondrial membrane potential of MCG-803 cells was decreased by 56.26%.CONCLUSION: 1386A has an inhibitory effect on the cell growth in MCG-803 cells through the mitochondrial pathway.     

Albumin overload induces α-smooth muscle actin expression in proximal tubular epithelial cells

AIM: To study whether albumin overload in proximal tubular epithelial cells (PTCs) induces epithelial to myofibroblast transdifferentiation (EMT). METHODS: Rat renal proximal tubular cell line NRK52E was cultured on 6 well plates. When the cells reached 70% confluens or complete confluens, cells were serum starved for 24 h. Different concentrations of delipidated bovine serum albumin (dBSA), ranging from 0-30 g/L, were then added to the cells. The media was changed every 48 h until the end of 144 h. Cell shapes were monitored by light microscope during experiment. Cell structures were detected by electron microscopy. Epithelial cell markers: E-cadherin, β-catenin, and myofibroblast marker: α-smooth muscle actin (α-SMA) were detected by immunofluorescent microscopy and Western blotting. RESULTS: dBSA overload induced the expression of α-SMA in sub-confluent NRK52E, and a few cells elongated, but the induced expression of α-SMA was not in a dose dependent manner. dBSA overload did not induce the expression of α-SMA in complete confluent NRK52E, cell shape did not change either. dBSA overload did not inhibit expression of E-cadherin or β-catenin both in sub-confluent and complete confluent NRK52E. The electron microscope showed that these cells retained epithelial phenotype, with microvilli and tight junction. CONCLUSION: Albumin overload induces PTCs expressing myofibroblast marker α-SMA and promotes EMT. However, complete EMT does not achieve. Complete confluens (cell-cell contacts) inhibits albumin induced α-SMA expression in PTCs.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Mesenchymal stem cells contribute to the formation of liver fibrosis

AIM: Myofibroblasts play key roles in the formation of liver fibrosis. It has been reported that bone marrow mesenchymal stem cells can differentiate into myofibroblasts, and migrate to the damaged organs to participate the fibrotic process. Therefore, this study was designed to investigate the possible function and mechanism of bone marrow mesenchymal stem cells in developing liver fibrosis. METHODS: A mesenchymal stem cell line Ap8c3 was tagged with enhanced green fluorescent protein(eGFP)(Ap8c3-eGFP). The rat model of liver fibrosis was established by bile duct ligation(BDL) or injection of pig serum(IPS). Ap8c3-eGFP was intravenously injected into BDL or IPS-induced liver fibrotic rats. The eGFP positive(eGFP⁺) cells and expression of α-smooth muscle actin(α-SMA) in these eGFP⁺ cells in rat livers and bone marrow(BM) were detected. RESULTS: Intravenous engraftment of Ap8c3- eGFP resulted in homing to fibrotic livers as well as BM. Co-expression of α-SMA by eGFP⁺ cells was found in liver sections, and eGFP⁺ cells were found mainly in fibrotic septum in fibrotic livers. Ap8c3-eGFP was observed to differentiate into myofibroblasts, which specifically expressed α-SMA after homing to bone marrow.CONCLUSION: Differentiation of mesenchymal stem cells into myofibroblasts plays important roles in the formation of liver fibrosis. BM-derived myofibroblasts can migrate to damaged livers and participate in the formation of liver fibrosis.     

Changes of RyR-mediated Ca2+ release in VSMCs is involved in the development of vascular hyporeactivity in hemorrhagic shock rats

AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca²⁺ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca²⁺ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca²⁺ concentration in VSMCs were evaluated by fura3-AM and the role of IP₃R and RyR mediated Ca²⁺ release from calcium store was further observed. The role of IP₃R and RyR mediated Ca²⁺ release from Ca²⁺ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca²⁺, NE upregulated by mobilizing Ca²⁺ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca²⁺ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca²⁺ releasing activator caffeine. However, the increase in triggered by IP₃R-mediated Ca²⁺ release agonist adenophostin A (10^-5 mol/L) and ATP-Na₂ (10^-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP₃R mediated Ca²⁺ release with ATP-Na₂ (10^-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP₃R mediated Ca²⁺ release with heparin (10⁴ U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca²⁺-free K-H solution, RyR antagonist ryanodine (10^-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10^-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca²⁺ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.     

Donor age affects confluent EPCs on phenotypic transition,proliferation and migration of smooth muscle cells

AIM: To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells (SMCs). METHODS: Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 (containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1×10⁵ units/L of penicillin and streptomycin, respectively). EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding. Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats. Vascular SMCs were cultured by tissue explant method and identified by α-SM-actin immunofluorescence. In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower chamber. The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE). The protein expression of α-SM-actin and osteopontin was detected by Western blotting. [³H]-TdR incorporation assay was used to determine the proliferation. SMC migration was analyzed by scratch wound healing assay. RESULTS: Compared with P3 group, α-SM-actin expression in P4 group significantly decreased and osteopontin protein expression obviously increased, whereas no significant change was found in P4YE group. Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs. Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phenotype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION: Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs proliferation and migration. Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.     

Double TdR blocking induces synchronization of cell cycle in human gastric cancer SGC-7901 cells

AIM: To investigate the effects of double thymidine deoxyribonucleoside (TdR) blocking on the cell cycle of human gastric cancer SGC-7901 cells.METHODS: SGC-7901 cells in the logarithm period were selected in the study and treated with TdR at concentration of 2 mmol/L, 4 mmol/L or 8 mmol/L for 15 h as the first blocking. After incubation in TdR-free medium for 10 h, the cells were treated with TdR at same concentrations again for another 15 h as the second blocking. The blocked cells were released by washing in fresh medium twice and incubation in TdR-free medium containing 10% fetal bovine serum for 12 h. The cells were collected and the cell cycle was detected by flow cytometry.RESULTS: By double TdR (2 mmol/L, 4 mmol/L and 8 mmol/L) blocking to synchronize the cell cycle, the cells in G₀/G₁ phase accounted for 77.3%, 77.5% and 77.0%, respectively. After further incubation for 12 h in TdR-free medium, the proportion of the cells in each phase of the cell cycles returned to normal range. CONCLUSION: The method of double TdR blocking is an ideal access in short term to acquire a large number of cells in G₀/G₁ phase.     

Effects of endothelin-1 on the induction of the transdifferentiation of human airway sub-epithelial fibroblasts

AIM: To explore the role of endothelin-1 (ET-1) in initiating transdifferentiation of sub-epithelial fibroblasts (SEFs) into myofibroblasts and its ionic and signal transduction mechanism.METHODS: Human SEFs or SEFs plated in collagen gels were co-cultured with human bronchial epithelial cells (16HBE) treated with lipopolysaccharide (LPS) plus mechanical scratch. ET receptor A inhibitor (BQ123) or the inhibitors specific for p38 MAPK, ERK1/2 were added, repectively. The expression of α-smooth muscle actin (α-SMA) in the SEFs and contractility of the collagen gels containing with SEFs as well as the effects of p38 MAPK or ERK1/2 on α-SMA expression were evaluated. Using Ca2+ sensitive Fluo-3/AM, dynamic changes of intracellular calcium concentration (［Ca²⁺］_i) were observed in the SEFs by laser confocal microscopy.RESULTS: Injured 16HBE induced the transdifferentiation of myofibroblasts, which expressd α-SMA and increased contractility. BQ123 blocked the induction to a certain extent. Injured 16HBE activated p38 MAPK and ERK1/2 pathways in SEFs, both inhibitors of p38 MAPK and ERK1/2 attenuated the induction of α-SMA by injured 16HBE. The addition of exogenous ET-1 enhanced α-SMA expression and activated p38 MAPK, ERK1/2 pathways in the SEFs. Additionally, ET-1 significantly facilitated Ca²⁺ inflow into the fibroblasts.CONCLUSION: Injured 16HBE induces the transdifferentiation of SEFs into myofibroblasts, which is involved in the activation of p38 MAPK and ERK1/2 pathways. The ET-induced influx of Ca²⁺ may be an early signal for initiating the myofibroblasts transdifferentiation.     

Impact of serial activation of embryonic pax2 and WT1 genes on transdifferentiation of renal tubular epithelial cells

AIM: To investigate the mechanisms of transdifferentiation of renal tubular epithelial cells by the observation of serial activation of embryonic pax2 and WT1 genes in chronic renal failure model of 5/6 nephrectomized injury. METHODS: Embryo of mice, cultured renal tubule cells and chronic renal failure rat model of 5/6 nephrectomized injury were established. The expressions of paired Box gene (pax2) and Willm’s tumor gene (WT1), as well as α-smooth muscle actin (α-SMA), a phenotype of mesenchymal cells, were detected by RT-PCR and immunohistochemistry techniques. RESULTS: (1) Expressions of pax2 and WT1 mRNA began at 11.5 d and 14 d of embryo and increased gradually, and expression in trace quantity at 2 weeks after birth. Immunohistochemistry analysis revealed that WT1 only expressed in the glomerular podocytes and expressions of pax2 and WT1 in adult renal tubular cells were negative. (2) Deno-expression of pax2 and WT1 in some tubular cells appeared at week 2 and peaked at week 4 in 5/6 nephrectomized rats, both showing a same trend of expression. However, pax2 showed another peak at week 10 afterwards. (3) Re-expressions of pax2 and WT1 in TEC at 0.5 and 24 h after treated with IL-1α (10 μg/L) or AngII (10-9 mol/L) were observed respectively, followed by upregulation of α-SMA expression, and mesenchymal cells characters were shown. The effects were inhibited by Ang II receptor 2 antagonist and WT1 antibody, respectively. CONCLUSION: Adult renal tubule cells acquire re-activation of embryonic pax2 and WT1 genes and phenotypes of mesenchymes when challenge with certain injuries. Deno-expression of pax2 and WT1 genes closely relates with high concentration of bioactive facors, such as AngII and IL-1, which are involved in the mechanisms of renal tubule cells transdifferentiation.     

Effects of adrenomedullin on proliferation of vascular smooth muscle cells induced by urotensin Ⅱ

AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [³H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-³²P]-ATP. RESULTS:UⅡ(10^-8mol/L) significantly increased [³H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10^-10,10^-9,10^-8mol/L ADM decreased [³H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.     

Pathogenesis of endothelial-mesenchymal transition in pulmonary arterial hypertension

Endothelial-mesenchymal transition (EndMT) is a biological process through which endothelial cells change their endothelial phenotype into a mesenchymal or myofibroblastic phenotype with the expression of mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin. When the pulmonary vascular endothelial cells are exposed to hypoxia and inflammatory stimulation, vascular smooth muscle cells in the outer and middle membrane accumulate through EndMT process, leading to pulmonary vascular remodeling and pulmonary arterial hypertension (PAH). Transforming growth factor β (TGF-β)/bone morphogentic protein (BMP) signaling pathways promote EndMT process by bone morphogentic protein receptor 2 (BMPR2) gene mutation which up-regulates high mobility group protein A1 (HMGA1) gene and contributes to protein expression such as Slug and Snail, thus resulting in PAH. In brief, TGF-β/BMP signaling pathways and related regulators play an important role in pulmonary vascular reconstruction and the formation of PAH.     

Blockage of core fucosylation contributes to inhibition of epithelial mesenchymal transition of renal tubular epithelial cells

AIM: To investigate the role of inhibiting core fucosylation in the process of epithelial mesenchymal transition (EMT) in HK-2 cells.METHODS: An EMT cell model with transforming growth factor-β₁(TGF-β₁) was established and RNAi technique was used to silence the expression of α-1,6-fucosyltransferase ( FUT8) gene which is responsible to catalysation of core fucose. The morphological changes of HK-2 cells were observed under light microscope. The epithelial cell marker E-cadherin and fibrotic cell markers N-cadherin, fibroblast-specific protein-1(FSP-1) and α-smooth muscle actin(α-SMA) were detected by Western blotting and immunocytochemistry. The apoptosis induced by TGF-β₁ was determined by flow cytometry. RESULTS: After incubated with TGF-β₁ at the concentration of 5 μg/L for 48 h, HK-2 cells lost epithelial morphology and showed fibrotic morphology. The expression of α-SMA, FSP-1 and N-cadherin was markedly increased, while E-cadherin was decreased. Meanwhile, the expression of FUT8 was up-regulated, and the apoptosis of the cells increased. However, pre-incubation of the cells with FUT8 siRNA inhibited these changes above.CONCLUSION: The core fucosylation involves in the process of EMT in HK-2 cells. Blockage of core fucosylation results in the inhibition of EMT in HK-2 cells.