Microvascular Endothelial Cells In Rats Research Articles

Regulation of Microtubule Stability in Pulmonary Microvascular Endothelial Cells in Rats with Severe Acute Pancreatitis: Qingyi Decoction is a Potential CDK5 Inhibitor.

Explore the therapeutic effects and regulatory mechanism of Qingyi Decoction (QYD) on severe acute pancreatitis (SAP) associated acute lung injury (ALI). We identified the constituents absorbed into the blood of QYD based on a network pharmacological strategy. The differentially expressed genes from the GEO database were screened to identify the critical targets of QYD treatment of SAP-ALI. The SAP-ALI rat model was constructed.Some methods were used to evaluate the efficacy and mechanism of QYD in treating SAP-ALI. LPS-stimulated pulmonary microvascular endothelial cell injury simulated the SAP-induced pulmonary endothelial injury model. We further observed the therapeutic effect of QYD and CDK5 plasmid transfection on endothelial cell injury. 18 constituents were absorbed into the blood, and 764 targets were identified from QYD, 25 of which were considered core targets for treating SAP-ALI. CDK5 was identified as the most critical gene. The results of differential expression analysis showed that the mRNA expression level of CDK5 in the blood of SAP patients was significantly up-regulated compared with that of healthy people. Animal experiments have demonstrated that QYD can alleviate pancreatic and lung injury inflammatory response and reduce the upregulation of CDK5 in lung tissue. QYD or CDK5 inhibitors could decrease the expression of NFAT5 and GEF-H1, and increase the expression of ACE-tub in SAP rat lung tissue. Cell experiments proved that QYD could inhibit the expression of TNF-α and IL-6 induced by LPS. Immunofluorescence results suggested that QYD could alleviate the cytoskeleton damage of endothelial cells, and the mechanism might be related to the inhibition of CDK5-mediated activation of NFAT5, GEF-H1, and ACE-tub. CDK5 has been identified as a critical target for pulmonary endothelial injury of SAP-ALI. QYD may partially alleviate microtubule disassembly by targeting the CDK5/NFAT5/GEF-H1 signaling pathway, thus relieving SAP-induced pulmonary microvascular endothelial cell injury.

Cigarette Smoke Extract and Lipopolysaccharide Induce Pyroptosis in Pulmonary Microvascular Endothelial Cells of Rats.

We studied the effect of cigarette smoke extract (CSE), LPS, or their combination on the activity and pyroptosis of pulmonary microvascular endothelial cells (PMVEC) in rats. PMVEC were cultured without treatment, with CSE in different concentrations (1-25%), with 20 ng/ml LPS, or with 20% CSE+20 ng/ml LPS. Cell viability was determined using the CCK8 kit, apoptosis was evaluated by flow cytometry, and cell morphology was evaluated using light microscopy. The content of IL-1β and IL-18 was measured by ELISA. CSE decreased cell viability in a dose-dependent manner. The morphology of cells in the CSE+LPS group showed the most significant cytomorphological changes and the highest pyroptosis rate. Flow cytometry showed that the apoptosis rates in the CSE and LPS groups were higher than in the control group, but the highest rate of apoptosis was revealed in the CSE+LPS group (p<0.01). The levels of IL-18 and IL-1β in the cell supernatant of the CSE, LPS, and CSE+LPS groups were significantly (p<0.01) increased in comparison with the control. These levels in the CSE+LPS group were higher (p<0.01) than in other groups. There were no differences between the CSE and LPS groups. Thus, the effect of CSE on cell viability is dose-dependent. Combined treatment with CSE+LPS can induce cell pyroptosis and increase the levels of inflammatory cytokines in PMVEC. These observations demonstrated that pyroptosis caused by CSE and LPS can play an important role in pulmonary vascular remodeling.

Expression of concern: Matrine inhibits hypoxia/reoxygenation-induced apoptosis of cardiac microvascular endothelial cells in rats via the JAK2/STAT3 signaling pathway [Biomedicine & Pharmacotherapy 106 (2018) 117–124

Expression of concern: Matrine inhibits hypoxia/reoxygenation-induced apoptosis of cardiac microvascular endothelial cells in rats via the JAK2/STAT3 signaling pathway [Biomedicine & Pharmacotherapy 106 (2018) 117–124

Retraction.

Retraction: "Effects of astragalus polysaccharide on apoptosis of myocardial microvascular endothelial cells in rats undergoing hypoxia/reoxygenation by mediation of the PI3K/Akt/eNOS signaling pathway," by Qingling Zhou, Guowei Meng, Fei Teng, Qiang Sun, Yongshan Zhang, J Cell Biochem. 2018; 806-816: The above article, published online on 28 June 2017 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.26243) has been retracted by agreement between the the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific contest. Thus, the editors consider the conclusions of this article to be invalid.

Preliminary study of icariin indicating prevention of steroid-induced osteonecrosis of femoral head by regulating abnormal expression of miRNA-335 and protecting the functions of bone microvascular endothelial cells in rats.

The pathogenesis of osteonecrosis of the femoral head (ONFH) is unclear. Our previous study demonstrated that upregulated miR-335 in bone microvascular endothelial cells (BMECs) might be associated with the disease of steroid-induced ONFH. Here, we study the preventive effect of ICA on steroid-induced ONFH in rats. 90 rats were separated into three groups: control group, methylprednisolone (MPS) group, and MPS + Icariin (ICA) group. Four weeks later, histological analyses were performed. Thrombomodulin (TM) and vascular endothelial growth factor (VEGF) were tested. MiRNA-335 expression was screened in the three groups using Agilent Gene Spring GX software. Target genes of miRNA-335 were detected by bioinformatics analysis. The functions of BMECs were analyzed by scratch, angiogenesis and cell survival rate. ICA can prevent the occurrence of steroid-associated ONFH in rats and reduce the amount of TM and VEGF in serum induced by glucocorticoids. ICA could regulate the overexpression of miRNA-335 induced by glucocorticoids. We predicted the Gene ontology (GO) and signaling pathways of target genes. At 24 hours, we found that ICA significantly promoted BMECs migration abilities. We also found that ICA could promote the angioplasty ability of BMECs. ICA could improve the survival rate of BMECs after steroid-induced injury. ICA is effective to prevent the occurrence of steroidinduced ONFH. ICA has a protective effect against steroid-induced BMECs injury. ICA regulated the imbalance of miRNA-335 expression induced by the glucocorticoid in BMECs, which provides a new viewpoint to explore the mechanism of ICA in preventing steroid-induced ONFH.

Effect of the Recombinant Adenovirus-Mediated HIF-1 Alpha on the Expression of VEGF in the Hypoxic Brain Microvascular Endothelial Cells of Rats.

ObjectiveTo investigate the effect of recombinant adenovirus-mediated HIF-1 alpha (HIF-1α) on the expression of vascular endothelial growth factor (VEGFA) and HIF-1α in hypoxic brain microvascular endothelial cells (BMEC) in rats.MethodsPrimary cultured rat BMEC in vitro were treated without or with either recombinant adenovirus-mediated hypoxia-inducible factor-1 alpha (AdHIF-1α) or recombinant adenovirus empty vector (Ad) in the presence of CoCl2 (simulating hypoxia conditions), or were grown under normoxia conditions. The expression of VEGFA and HIF-1α was analyzed at 12h, 24h, 48h and 72h incubation time, respectively. We also accessed a GEO dataset of stroke to analyze in vivo the alteration of HIF-1α and VEGFA expression, and the correlations between HIF-1α, VEGFA and CD31 mRNA levels in vascular vessels after stroke.ResultsVEGFA and HIF-1α expression were significantly higher in at each time point in the AdHIF-1α than other groups (p<0.05), whereas the Ad group and hypoxia group, showed no statistically significant difference (p>0.05). Moreover, VEGFA and HIF-1α levels were significantly higher in BMEC under hypoxia conditions than normoxia conditions (p <0.05). Both HIF-1α and VEGFA expression significantly increased after stroke in vivo with 1.30 and 1.57 fold-change in log2, respectively. There were significantly positive associations between HIF-1α, VEGFA and CD31 mRNA levels in vivo after stroke.ConclusionHypoxia-induced HIF-1α and VEGFA expression in vascular vessels, and recombinant AdHIF-1α could up-regulate VEGFA, and enhance HIF-1ααlevels in BMEC in vitro, which may play an important role in the recovery of stroke.

RETRACTED: Matrine inhibits hypoxia/reoxygenation-induced apoptosis of cardiac microvascular endothelial cells in rats via the JAK2/STAT3 signaling pathway

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. An Expression of Concern for this article was previously published while an investigation was conducted (see related editorial: https://doi.org/10.1016/j.biopha.2022.113812). This retraction notice supersedes the Expression of Concern published earlier. Concerns were raised about the provenance of the flow cytometry data shown in Figure 5A, as detailed here: https://pubpeer.com/publications/46C8B5439C5C617A60494BA4C15479; and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. Independent analysis also identified additional suspected image duplications between the Bax and Bcl-2 Western blots in Figure 6A. The journal requested the corresponding author comment on these concerns and provide the associated raw data. The authors did not respond to this request and therefore the Editor-in-Chief decided to retract the article.

GW28-e1067 Urotensin II induces endothelial-mesenchymal transition of cardiac microvascular endothelial cells via endoplasmic reticulum stress

Recent studies suggest that endothelial-mesenchymal transition (EndMT) as a link between endoplasmic reticulum stress (ER stress) and fibrosis. We previous found that urotensin II (UII) could promote EndMT in cardiac microvascular endothelial cells of rats. Therefore, in the present study, we

Role of phosphorylated Moesin in the injury of pulmonary microvascular endothelial cells of rats and its mechanism

To investigate the role of phosphorylated Moesin (p-Moesin) in the injury of pulmonary microvascular endothelial cells (PMVECs) of rats induced by tumor necrosis factor-α (TNF-α), and to approach the impact of Rac1 signal pathway on Moesin phosphorylation. PMVECs of rats were cultured in vitro and passed on to the third generation, and the TNF-α time-effect experiment, dose-effect experiment and Rac1 signaling pathway intervention experiment were performed respectively. (1) Time-effect experiment: PMVECs were stimulated with 10 μg/L TNF-α for 0, 15, 30 minutes and 1, 3, 6, 12 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. (2) Dose-effect experiment: PMVECs were stimulated with 0, 0.1, 1, 10 μg/L TNF-α for 6 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. (3) Rac1 signaling pathway intervention experiment: PMVECs were divided into two parts, which were pretreated with 3 mL Rac1 specific inhibitor NSC23766 (200 μmol/L) for 0.5 hour or Rac1 specific agonist O-Me-cAMP (200 μmol/L) for 1 hour, respectively, and then incubated with 10 μg/L TNF-α for 6 hours. The PMVECs without treatment were served as blank control group, and those were treated with only O-Me-cAMP, NSC23766 or TNF-α were served as corresponding groups. The protein expressions of Moesin and p-Moesin were determined by Western Blot. (1) Time-effect experiment results: the expression of Moesin showed no change among all time points after 10 μg/L TNF-α stimulated PMVECs. But the expression of p-Moesin was sharply up-regulated at 15 minutes after TNF-α stimulation as compared with 0 minute (p-Moesin/Moesin: 4.399±0.523 vs. 1.000±0.195), peaked at 30 minutes (6.069±0.557), and then gradually decreased after 1 hour (5.005±0.544, 4.599±0.478, 1.742±0.288, 1.503±0.352 at 1, 3, 6, 12 hours, respectively) with significant difference among all time points (F = 15.397, P = 0.002). (2) Dose-effect experiment results: no significant change in expression of Moesin was found among all doses of TNF-α incubated with PMVECs for 6 hours. But the expression of p-Moesin was significantly up-regulated after TNF-α stimulation with 0.1 μg/L as compared with 0 μg/L (p-Moesin/Moesin: 2.194±0.430 vs. 1.000±0.273), which showed an upward trend with dose increase in TNF-α (3.201±0.688 and 4.413±0.296 with 1 μg/L and 10 μg/L TNF-α, respectively) with significant difference among all doses (F = 92.513, P < 0.001). (3) Rac1 signaling pathway intervention experiment results: there was no significant difference in Moesin expression among all the groups. Compared with blank control group, Rac1 specific agonist O-Me-cAMP or Rac1 specific inhibitor NSC23766 alone could not change the expression of p-Moesin, while TNF-α could induce p-Moesin expression. Compared with TNF-α group, the expression of p-Moesin induced by TNF-α was up-regulated by NSC23766 (p-Moesin/Moesin: 2.612±0.355 vs. 1.911±0.297, P < 0.05), and it was attenuated by O-Me-cAMP (p-Moesin/Moesin: 1.928±0.331 vs. 3.030±0.353, P < 0.05). The phosphorylation of Moesin is involved in the damage of TNF-α-induced PMVECs in rats, and PMVECs damage could be alleviated by modulating Moesin phosphorylation in the Rac1 signaling pathway.

Effects of astragalus polysaccharide on apoptosis of myocardial microvascular endothelial cells in rats undergoing hypoxia/reoxygenation by mediation of the PI3K/Akt/eNOS signaling pathway.

The study explores the effect of astragalus polysaccharide (APS) mediating P13K/Akt/eNOS signaling pathway on apoptosis of myocardial microvascular endothelial cells (MMECs) in hypoxia/reoxygenation (H/R). MMECs were classified into blank, H/R, H/R + 25 mg/L APS, H/R + 50 mg/L APS, H/R + 100 mg/L APS, H/R + LY, and HR + 100 mg/L APS + LY groups. Cell viability was detected using MTT assay and apoptotic cell morphological changes by Hoechst staining. NO content, cell cycle and apoptosis, PI3K/Akt/eNOS signaling pathway proteins were detected using nitrate reductase assay, flow cytometry and Western blotting. An increased cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins, and a decreased apoptosis rate was observed in the H/R + 50 mg/L APS and H/R + 100 mg/L APS groups compared with the H/R and H/R + 25 mg/L APS groups. Compared with the H/R + 50 mg/L APS group, the apoptosis rate decreased, whereas the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins increased in the H/R + 100 mg/L APS group. The H/R + LY and HR + 100 mg/L APS + LY groups followed opposite trends. In comparison to the HR + 100 mg/L APS group, the apoptosis rate in the H/R + LY and HR + 100 mg/L APS + LY groups increased, and the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins decreased. Collectively, APS improves the damage caused by H/P by mediating PI3K/Akt/eNOS signaling pathway.

Inhibitory effects of PGA1 and TRI on the apoptosis of cardiac microvascular endothelial cells of rats.

The present study investigated the protective effects and molecular mechanism of prostaglandin A1 (PGA1) and triptolide (TRI) on apoptosis of cardiac microvascular endothelial cells (CMVECs) in rats. CMVECs of rats were isolated and then cultured. MTT method was used to select and establish a cell hypoxia reoxygenation cell model. The cells were divided into four groups: Normoxia control group (C, normal oxygen), hypoxia reoxygenation group (H/R, hypoxia for 12 h/reoxygenation for 6 h), PGA1 group (H/R+PGA1) and TRI group (H/R+TRI). The growth of cells in each of the group was observed. B-cell lymphoma 2 (Bcl-2) mRNA expression in CMVECs and expression of Bcl-2 mRNA after PGA1 and TRI treatment were determined by RT-PCR. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Bcl-2 mRNA decreased significantly after hypoxia stimulation of CMVECs of rats. The expression of Bcl-2 mRNA was significantly higher in comparison to hypoxia stimulation group after treatment with PGA1 and TRI (P<0.01). The elevated effect of PGA1 on Bcl-2 mRNA was stronger than that of the TRI group (P<0.05). The number of CMVECs reduced significantly after hypoxia. By contrast, DNA fragmentation and the number of endothelial cell apoptosis were increased significantly. However, Bcl-2 mRNA expression decreased significantly, after PGA1 and TRI treatments. Furthermore, the number of apoptotic cells reduced and Bcl-2 mRNA expression increased (P<0.01). PGA1 and TRI significantly upregulated the expression of Bcl-2 mRNA, inhibited the activation of CMVECs and were able to achieve the protective effect on apoptosis of CMVECs in hypoxia-oxygenated rats.

Rhubarb Antagonizes Matrix Metalloproteinase-9-induced Vascular Endothelial Permeability.

Background:Intact endothelial structure and function are critical for maintaining microcirculatory homeostasis. Dysfunction of the latter is an underlying cause of various organ pathologies. In a previous study, we showed that rhubarb, a traditional Chinese medicine, protected intestinal mucosal microvascular endothelial cells in rats with metastasizing septicemia. In this study, we investigated the effects and mechanisms of rhubarb on matrix metalloproteinase-9 (MMP9)-induced vascular endothelial (VE) permeability.Methods:Rhubarb monomers were extracted and purified by a series of chromatography approaches. The identity of these monomers was analyzed by hydrogen-1 nuclear magnetic resonance (NMR), carbon-13 NMR, and distortionless enhancement by polarization transfer magnetic resonance spectroscopy. We established a human umbilical vein endothelial cell (HUVEC) monolayer on a Transwell insert. We measured the HUVEC permeability, proliferation, and the secretion of VE-cadherin into culture medium using fluorescein isothiocyanate-dextran assay, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, and enzyme-linked immunosorbent assay, respectively, in response to treatment with MMP9 and/or rhubarb monomers.Results:A total of 21 rhubarb monomers were extracted and identified. MMP9 significantly increased the permeability of the HUVEC monolayer, which was significantly reduced by five individual rhubarb monomer (emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein) or a combination of all five monomers (1 μmol/L for each monomer). Mechanistically, the five-monomer mixture at 1 μmol/L promoted HUVEC proliferation. In addition, MMP9 stimulated the secretion of VE-cadherin into the culture medium, which was significantly inhibited by the five-monomer mixture.Conclusions:The rhubarb mixture of emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein, at a low concentration, antagonized the MMP9-induced HUVEC monolayer permeability by promoting HUVEC proliferation and reducing extracellular VE-cadherin concentrations.

Effect of bilirubin on proliferation of pulmonary microvascular endothelial cells of rats

Objective To evaluate the effect of bilirubin on proliferation of pulmonary microvascular endothelial cells (PMVECs) of rats.Methods Primary PMVECs harvested from 10 healthy male Sprague-Dawleyrats,weighing 120-150 g,aged 2-3 months,were cultured,purified and identified.PMVECs were seeded in low-glucose DMEM culture medium or 96-well culture plates,and divided into 5 groups according to the random number table:control group (group C) and different concentrations of bilirubin groups (B1-4 groups) with 30 wells or48 flasks in each group.In group C,low-glucose DMEM 1 ml was added.In B1-4 groups,5,10,20 and 50 μmol/L bilirubin 1 ml were added,respectively.At 24,48 and 72 h of incubation,proliferation of PMVECs was measured using CCK-8 assay and 3 H-TdR incorporation assay,Akt1 mRNA and Smad3 mRNA expression was measured by RT-PCR,and phosphor-Akt1 (p-Akt1) protein and Smad3 protein expression was detected using Western blot.Results Compared with group C,no significant changes were found in each parameter mentioned above at each time point in B1 and B2 groups,the proliferation of PMVECs was weakened,and Akt1 mRNA,p-Akt1 protein and Smad3 protein and mRNA expression was down-regulated at 48 h of incubation in B3 group,and the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 48 and 72 h of incubation in B4 group.Compared with group B3,the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 72 h of incubation in B4 group.Conclusion High concentration of bilirubin can inhibit proliferation of PMVECs and down-regulated expression of Akt1 and Smad3 is involved in the mechanism. Key words: Bilirubin; Cell proliferation; Endothelial cells

Effect of tumor necrosis factor-α on expression of interleukin-17 receptor C in pulmonary microvascular endothelial cells in rats

To explore the effects of tumor necrosis factor-alpha (TNF-α) or methylprednisolone on the expression of interleukin-17 receptor C (IL-17RC) in rat pulmonary microvascular endothelial cells (RPMVEC). Culture RPMVEC were randomly divided into dose-dependent and time-dependent groups. In dose-dependent group, cells were cultured with TNF-α (0, 0.1, 1, 10 µg/L TNF-α) for 3 h. In time-dependent group, cells were cultured with TNF-α (10 µg/L) for 0, 1, 3, 6, 12 h. In the drug intervention group, cells were cultured with TNF-α (10 µg/L) or methylprednisolone (200 µg/L) +TNF-α (10 µg/L) for 3 h respectively. The expression of IL-17RC in isolated and cultured RPMVEC was identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry. The changes of IL-17RC mRNA were detected by RT-PCR after the stimulation of RPMVEC by TNF-α or methylprednisolone. RT-PCR and Western blot revealed that IL-17RC mRNA and protein were present in RPMVEC. The product of IL-17RC immunocytochemical reaction was predominantly located in cytoplasm and cytomembrane. In RPMVEC TNF-α significantly up-regulated IL-17RC mRNA in a dose-dependent manner (0 µg/L TNF-α group: 0.241 ± 0.010, 0.1 µg/L TNF-α group: 0.372 ± 0.017, 1 µg/L TNF-α group: 0.452 ± 0.017, 10 µg/L TNF-α group: 0.643 ± 0.042, F = 33.774, P < 0.05). In time-dependent group, the expression of IL-17RC mRNA rose at 1 h (0.417 ± 0.038), peaked at 3 h (0.674 ± 0.018), then decreased gradually at 6 h (0.378 ± 0.035), but stayed higher at 12 h (0.318 ± 0.032). When compared with 0 h group (0.197 ± 0.008), there were significant differences (F = 37.903, P < 0.05). Methylprednisolone caused a marked attenuation of TNF-α-induced IL-17RC expression (0.333 ± 0.031 vs 0.660 ± 0.026, F = 89.637, P < 0.05). IL-17RC is predominantly present in cytomembrane and cytoplasm of RPMVEC. TNF-α up-regulates the expression of IL-17RC mRNA. Methylprednisolone inhibits the elevated expression of IL-17RC mRNA induced by TNF-α so as to relieve the inflammatory response of PMVEC.

Changes in expression of transforming growth factor beta-1 during myogenic differentiation of pulmonary microvascular endothelial cells in rats with hepato-pulmonary syndrome

Objective To investigate the changes in the expression of transforming growth factor beta-1 (TGF-β1) during differentiation of pulmonary microvascular endothelial ceils (PMVECs) into smooth muscle cells in rats with hepato-pulmonary syndrome (HPS).Methods Primary PMVECs were harvested from healthy adult SD rats of both sexes aged 3-4 months and inoculated in low-glucose DMEM culture medium (1(6/cm2 ) and randomly divided into 2 groups ( n =24 dishes each):control group ( group C) and HPS group.HPS was produced by chronic ligation of common bile duct.In group C serum obtained from normal rats was added to PMVECs,while in HPS group serum obtained from rats with HPS was added.The final concentration of serum was 10％.After being incubated for 24,48 and 72 h,the expression of SM-MHC,SM-α-actin and calponin protein and TGF-β1 mRNA and protein in PMVECs was determined by RT-PCR and Western blot analysis.Results The expression of SMMHC,SM-α-actin and calponin protein.was positive in HPS group whereas the expression of SM-α-actin and calponin protein was negative and the expression of SM-MHC protein was barely detectable in group C.The expression of SM-MHC,TGF-β1 mRNA and protein was significantly higher in HPS group than in group C.The expression of SM-MHC,SM-α-actin and calponin protein and TGF-β1 mRNA and protein was increasing with duration of incubation from T1 to T3 in group HPS.Conclusion TGF-β1 plays an important role in the myogenic differentiation of PMVECs in rats with HPS. Key words: Hepatopulmonary syndrome; Endothelial cells; Lung; Transforming growth factor betal ; Cell differentiation

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague-Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil-ac-LDL (1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non-treated group; three ghrelin dosage groups (1×10-9, 1×10-8, 1×10-7 mol/l) and one ghrelin+PI3K inhibitor group (1×10-7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10-8 mol/l (0.271±0.041 compared with 0.199±0.021, P = 0.03) and 10-7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin-mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P = 0.15). At a concentration between 10-8 and 10-7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P = 0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P = 0.32). Ghrelin treatment significantly enhanced NO production in a dose-dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P = 0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin-stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P = 0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.

Expression and significance of fgl2 prothrombinase in cardiac microvascular endothelial cells of rats with type 2 diabetes

Microthrombosis may be involved in the pathogenesis of cardiac microangiopathy due to diabetes. Recent studies have shown that fibrinogen-like protein 2 (fgl2) plays a pivotal role in microthrombosis in viral hepatitis, acute vascular xenograft rejection and cytokine-induced fetal loss syndrome. The current study was designed to examine the expression of fgl2 in microvascular endothelial cells and investigate the effects of microthrombi due to fgl2 on cardiac function and structure in rats with type 2 diabetes. Following induction of type 2 diabetes, 24 rats were observed dynamically. Fgl2 expression and related cardiac microthrombosis were examined. Local or circulating TNF-α was measured. Coronary flow (CF) per min was calculated as an index of cardiac microcirculation. Cardiac function and morphology were evaluated. It was found that Fgl2 was highly expressed in cardiac microvascular endothelial cells of rats with type 2 diabetes, which was promoted by local or circulating TNF-α. The Fgl2 expression was associated with cardiac hyaline microthrombosis. In parallel with the fgl2 expression, CF per min, cardiac diastolic or systolic function and cardiac morphology were aggravated to some extent. It was concluded that in rats with type 2 diabetes, microthrombosis due to fgl2 contributes to the impairment of cardiac diastolic or systolic function and morphological changes.

Effect of shock lymph on the expression of inflammatory mediators in pulmonary micro-vascular endothelial cells of rats

To observe the effect of lymph collected during shock on free radical and expressions of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) mRNA of pulmonary micro-vascular endothelial cells (PMVECs) of rats in order to explore the mechanisms of damaging effect of lymph collected during shock to the PMVECs. PMVECs were isolated and cultured, and used at passage 3. The model of serious hemorrhagic shock was reproduced by maintaining the arterial blood pressure of rats at 40 mm Hg (1 mm Hg=0.133 kPa) for 90 minutes by exsanguination under aseptic condition. Mesentery lymph and portal vein blood were obtained from both shock rats and normal rats. PMVECs were respectively incubated in them for 6 hours, and at the same time, fetal bovine serum (FBS) and serum-free DMEM were used as culture media for comparison. The expressions of inducible nitric oxide synthase (iNOS), TNF-alpha and IL-6 mRNA were detected by the method of reverse transcription-polymerase chain reaction (RT-PCR), and the content of malondialdehyde (MDA), NO, TNF-alpha and IL-6 in culture supernatants were determined. After the PMVECs was treated by shock lymph at a final concentration of 4% for 6 hours, the expressions of iNOS, TNF-alpha and IL-6 mRNA in PMVECs and the contents of MDA, NO, TNF-alpha and IL-6 in culture supernatant fluids in shock lymph group were significantly increased compared with those of FBS group, normal lymph group, shock plasma group, normal plasma group and DMEM group. At the same time, the expressions of iNOS, TNF-alpha and IL-6 mRNA in PMVECs and the contents of NO in culture supernatant fluid of shock plasma group were significantly increased compared with those of FBS group, normal lymph group, normal plasma group and DMEM group (P<0.05 or P<0.01). The results demonstrate that the shock lymph in final concentration of 4% could enhance the expressions of iNOS, TNF-alpha and IL-6 of PMVECs, reduce the free radical, and as a result, induce damage to PMVECs.

P–185 - Transport mechanism of histidine by cultured lung microvascular endothelial cells in rats: Its enhancement by zinc.

P–185 - Transport mechanism of histidine by cultured lung microvascular endothelial cells in rats: Its enhancement by zinc.