The Expression of Estrogen Receptor is Dependent on the Estrogen Level and Associated with Cholesterol - Rich Diet in Female Rat''''s Heart and Vascular Endothelial Cells

Objective To study the effects of estrogen level and cholesterol - rich diet on the expression of estrogen receptor (ER) in cardiovascular tissues including vascular endothelial cells (VEC) of female rats. Methods The receptor binding assay (RBA) was adopted to measure the estrogen receptor level in aortic wall, heart and vascular endothelial cells of female rats on a cholesterol - rich diet. A ra-dioimmunoassay was employed to measure the level of serum estradiol. Results The number of ER significantly decreased in hearts, aorta and vascular endothelial cells in the ovariectomized rats and the rats on a cholesterol - rich diet. In contrast, the administration of estrogen somewhat restored the expression of ER. Conclusions For female rats, the level of estrogen affects the expression of ER in cardiovascular system. The number of ER decreases along with the decrease in the level of estrogen. A cholesterol - rich diet also can decrease the expression of ER in cardiovascular system of female rats.     

The Expression of Estrogen Receptor is Dependent on the Estrogen Level and Associated with Cholesterol- Rich Diet in Female Rat＇s Heart and Vascular Endothelial Cells

Objective To study the effects of estrogen level and cholesterol - rich diet on the expression of estrogen receptor (ER) in cardiovascular tissues including vascular endothelial cells (VEC) of female rats. Methods The receptor binding assay (RBA) was adopted to measure the estrogen receptor level in aortic wall, heart and vascular endothelial cells of female rats on a cholesterol - rich diet. A radioimmunoassay was employed to measure the level of serum estradiol. Results The number of ER significantly decreased in hearts, aorta and vascular endothelial cells in the ovariectomized rats and the rats on a cholesterol- rich diet. In contrast, the adminis-tration of estrogen somewhat restored the expression of ER. Conclusions For female rats, the level of estrogen affects the expression of ER in cardiovascular system. The number of ER decreases along with the decrease in the level of estrogen. A cholesterol -rich diet also can decrease the expression of ER in cardio-vascular system of female rats.     

Effects of Angiotensin Ⅱ and Valsartan on the Expression of ATR1/ATR2 Receptors,eNOS in Vascular Endothelial Cells

Background Angiotensin Ⅱ type 1 receptor (ATR1) / Angiotensin Ⅱ type 2 receptor (ATR2) usually interact with each other in their expression and physiological functions, and nitric oxide (NO) is always involved in ATR1 / ATR2 regulation in vivo. Endothelial cells play a crucial role in the maintenance of vascular function and in the prevention of cardiovascular diseases. Objectives To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) and ATR1 blocker valsartan on ATR1, ATR2 expression and their relation with endothelial nitric oxide synthase (eNOS) expression, and NO production in cultured vascular endothelial cells. Methods Human umbilical vein endothelial cell line (HUVEC) and bovine aortic endothelial cell (BAEC) were used. BAEC were isolated from aorta of newborn calf by enzyme digestion and cells of 3-5 passages were used. Cells were incubated with vehicle, Ang Ⅱ, valsartan, or Ang Ⅱ plus valsartan respectively for various periods. ATR1, ATR2, eNOS expression and NO production were detected. Results Incubation with AngⅡ or valsartan apparently downregulated ATR1 mRNA and protein expression in vascular endothelial cells, and the combination effect of the two drugs were more apparent. Ang Ⅱ showed a transient slightly promotive effect on eNOS and NO generation in BAEC and an apparently inhibitory effect with prolonged incubation, while valsartan can apparently reverse those effects. Conclusions Both Ang Ⅱ and valsartan downregulated the expression of ATR1 in vascular endothelial cells. The synergistic effect of the two drugs was more apparent. Prolonged incubation with Ang Ⅱ can apparently inhibit eNOS expression and NO production in endothelial cells, while valsartan can apparently reverse that inhibitory effect.     

Increased endothelin receptor B and G protein coupled kinase-2 in the mesentery of portal hypertensive rats

AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. RESULTS: PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31+ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA+ smooth muscle cells of arterioles and in the CD31+ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. CONCLUSION: Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.     

Gender differences in vascular reactivity of mesenteric arterioles in portal hypertensive and non-portal hypertensive rats

BACKGROUND Portal hypertension(PHT) is primarily caused by an increase in resistance to portal outflow and secondarily by an increase in splanchnic blood flow. Vascular hyporeactivity both in systemic circulation and in the mesenteric artery plays a role in the hyperdynamic circulatory syndrome.AIM To explore gender differences and the role of endogenous sex hormones in PHT and vascular reactivity of mesenteric arterioles in rats.METHODS Cirrhosis and PHT were established by subcutaneous injection of carbon tetrachloride(CCl_4) in both male and female integral and castrated rats(ovariectomized [OVX] in female rats, orchiectomy [ORX] in male rats). The third-order branch of the mensenteric artery was divided and used to measure vascular reactivity to vasoconstrictors.RESULTS No significant difference in portal pressure was observed between integral and castrated male PHT rats(15.2 ± 2.1 mm Hg vs 16.7 ± 2.7 mm Hg, P 0.05). The portal pressure in integral female PHT rats was lower than that in OVX female PHT rats(12.7 ± 2.7 mm Hg vs 16.5 ± 2.4 mm Hg, P 0.05). In PHT rats, the concentration response curves of the mesenteric arterioles to norepinephrine were shifted to the right, and the maximal responses(E_(max)) values were decreased and effective concentrations causing half maximum responses(EC_(50)) values were increased, compared to those of non-PHT rats, both in male and female rats.Compared to non-PHT integral male rats, the sensitivity of the mesenteric arterioles of non-PHT ORX male rats to norepinephrine was decreased(P 0.05).However, there was no difference between integral and ORX male rats with PHT.In integral female PHT rats, the concentration response curves were shifted to the left(P 0.05), and the E_(max) values were increased and EC_(50) values were decreased compared to OVX female PHT rats.CONCLUSION Clear gender differences were observed in mesenteric vascular reactivity in CCl_4-induced cirrhotic and PHT rats. Conservation of estrogen can retain the sensitivity of the mesenteric arterioles to vasoconstrictors and has a protective effect on splanchnic vascular function in PHT.     

Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NE-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection.In this study, we use pCMV-IκBαM vector to inhibit NE-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells.METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay.Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-I~BczMin in hibition of T cells adhesion to endothelial cells.RESULTS: We could find that NF-~B activity is inhibited by over-expression of non-degraded IκBα protein.Expression of adhesion molecules like ICAM-1, VCAM-1,and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector.CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NF-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection. In this study, we use pCMV-IκBαM vector to inhibit NF-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay. Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. RESULTS: We could find that NF-κB activity is inhibited by over-expression of non-degraded IκBα protein. Expression of adhesion molecules like ICAM-1, VCAM-1, and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector. CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Effects of ethanol on the properties of platelets and endothelial cells in model experiments

AIM: To investigate effects of ethanol on activity markers of atherosclerosis in an in vitro endothelial cell model. METHODS: After 24 h incubation with ethanol (0.0095%), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide, and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets, and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urokinase plasminogen activator receptor (uPAR), and membrane-type 1 matrix metalloproteinase (MT1-MMP) on endothelial cells were measured by flow cytometry. RESULTS: The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through pre-incubation with ethanol (P<0.05). Furthermore, platelets in direct contact with ethanol and with endothelial cells pre-incubated in ethanol showed a significant reduction in their CD40L expression (P<0.05). Ethanol had no significant effect on ICAM-1 and MT1-MMP expression on endothelial cells. CONCLUSION: Ethanol directly attenuates platelet activation and has significant endothelial cell-mediated effects on selected markers of atherosclerosis in vitro . These findings underline possible protective effects of ethanol on atherosclerosis.     

Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

AIM: To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. METHODS: MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell (HUVEC) monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocking studies using anti CD44 monoclonal antibodies or by hyaluronan digestion. RESULTS: Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice variants CD44v4, CD44v5, and CD44v7 were all up-regulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CONCLUSION: CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.     

Expression of gonadotropin-releasing hormone receptor and effect of gonadotropin-releasing hormone analogue on proliferation of cultured gastric smooth muscle cells of rats

AIM:To investigate the expression of gonadotropin-releasinghormone(GnRH)receptor and the effects of GnRH analog(alarelin)on proliferation of cultured gastric smooth musclecells(GSMC)of rats.METHODS:Immunohistochemical ABC methods and in situhybridization methods were used to dectect protein andmRNA expression of GnRH receptor in GSMC,respectively.Techniques of cell culture,OD value of MTT test,measureof ~3H-TdR incorporation,average fluorescent values ofproliferating cell nuclear antigen(PCNA)and flow oltometricDNA analysis were used in the experiment.RESULTS:The cultured GSMC of rats showed immunoreactivityfor GnRH receptor;positive staining was located in cytoplasm.GnRH receptor mRNA hybridized signals were also detectedin cytoplasm.When alarelin(10~(-9),10~(-7),10~(-5) mol/L)wasadministered into the medium and incubated for 24h,ODvalue of MTT,~3H-TdR incorporation and average fluorescentvalues of PCNA all decreased significantly as compared withthe control group(P<0.05).The maximum inhibitory effect oncell proliferation was achieved a concentration of 10~(-5) mol/Land it acted in a dose-dependent manner.Flow cytometricDNA analysis revealed that alarelin could significantlyenhance ratio of G_1 phase and decrease ratio of S phase ofGSMC of rats(P<0.05).The maximum inhibitory effect onratio of S phase was at the concentration of 10~(-5) mol/L andalso acted in a dose-dependent manner.CONCLUSION:Our data suggest that GnRH receptor canbe expressed by GSMC of rats.GnRH analogue can directlyinhibit proliferation and DNA synthesis of rat GSMC throughGnRH receptors.     

Glytan decreases portal pressure via mesentery vasoconstriction in portal hypertensive rats

AIM: To investigate the effects of Glytan on splanchnic hemodynamics and its reduction of portal pressure in portal hypertensive rats.METHODS:Glytan(Ganluotong in Chinese),is composed of salvianolic acid B and diammonium glycyrrhizinate.Portal hypertension(PHT)was induced in the rats by common bile duct ligation(BDL).Hemodynamic studies were performed using the colored microsphere method.Radioimmunoassay(RIA)was used to determine endothelin(ET)-1 levels in the mesenteric circulation.Western blotting methods were used to investigate the effect of Glytan on ET A receptor(ETAR),ET B receptor(ETBR),endothelial NO synthase(e NOS),G-protein-coupled receptor kinase(GRK)2,and β-arrestin 2 expression in the mesentery.The m RNA of ETAR and ETBR was determined using real-time polymerase chain reaction.RESULTS:Treatment with Glytan reduced portal pressure(PP)and portal territory blood flow(PTBF)and increased both mean arterial pressure(MAP)and splanchnic vascular resistance(SVR).Especially at 4wk,PP decreased by about 40%,while MAP increased by 13%,SVR increased by 12%,and PTBF decreased by about 21%.The effect of blood flow reduction was greatest in the mesentery(about 33%)at 4 wk.The mesenteric circulation ET-1 levels of BDL rats were lower and negatively correlated with PP at 4 wk.Glytan can increase mesenteric ET-1 content and inhibit ETBR,e NOS,GRK2,andβ-arrestin 2 expression in the mesentery.Moreover,Glytan showed no effect on the expression of ETAR protein and m RNA.CONCLUSION:The decreased PP and PTBF observed after Glytan treatment were related to increased mesenteric vasoconstriction and increased receptor sensitivity to vasoconstrictor.     

Mechanism of induction of fibroblast to corneal endothelial cell

Objective:To explore mechanism of nduction of fibroblast to corneal endothelial cell.Methods:Rabbit conjunctiva fibroblasts were used as feeder cells,rabbit oral mucosa epithelial cells were used as seed cells,and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium.The transformation effect was observed.Results:As concentration of mitomycin C increased,cell survival rale gradually decreased,cell proliferation was obviously inhibited when concentration ≥25μg/mL;5 days alter being treated by 5μg/mL.mitomycin C,cell body was enlarged and extended without cell fusion,however after being treated by 0.5 μg/mL mitomycin C,cell body was significantly proliferated and gradually fused:alter 3 weeks of culture,stratified epithelium appeared on rabbit oral mucosa epithelial cells,differentiation layers were 4-5 and were well differentiated,the morphology was similar to corneal endothelial cells;Under electron microscope,surface layer of cells were polygonal,tightly connected to another with microvilli on the bonier,there was hemidesmosome between basal cells and human denuded amniotic membrane.Conclusions:Fibroblast cells have the potential of multi-directional differentiation,effective induction can promote emergence of intercellular desmosomes between seed cells and emergence ol epithelial surface microvilli,and differentiate to the corneal endothelial cell.However,clinical application still needs more research and safetv evaluation.     

Expression of sialyl Lewis(a) relates to poor prognosis in cholangiocarcinoma

AIM: High levels of serum sialyl Lewisa (sLea) are frequently found in cholangiocarcinoma (CCA) patients and have been suggested to be a serum marker for CCA. However, the significance of this antigen in CCA is unknown. In this study, the clinical significance of sLea expression in CCA tissues and the possible role of sLea in vascular invasion in vitro were elucidated. METHODS: Expression of sLea in tumor tissues of 77 patients with mass-forming CCA and 33 with periductal infiltrating CCA was determined using immunohistochemistry. The in vitro assays on adhesion and transmigration of CCA cells to human umbilical vein endothelial cells were compared between CCA cell lines with and without sLea expression. RESULTS: sLea was aberrantly expressed in 60% of CCA tumor tissues. A significant relationship was found between the frequency of sLea expression and the mass-forming type CCA (P= 0.041), well differentiated histological grading (P=0.029), and vascular invasion (P=0.030). Patients with positive sLea expression had a significantly poorer prognosis (21.28 wk, 95% CI=16.75-25.81 wk) than those negative for sLea (37.30 wk, 95% CI=27.03-47.57 wk) (P<0.001). Multivariate analysis with adjustment for all covariates showed that patients positive for sLea possessed a 2.3-fold higher risk of death than patients negative for sLea (P<0.001). The role of sLea in vascular invasion was demonstrated using in vitro adhesion and transmigration assays. KKU-M213, a human CCA cell-line with a high expression of sLea, adhered and transmigrated to IL-1β-activated endothelial cells of the human umbilical vein more than KKU-100, the line without sLea expression (P<0.001). These processes were significantly diminished when the antibodies specific to either sLea or E-selectin were added to the assays (P<0.001) CONCLUSION: This study demonstrates the clinical significance of sLea expression in vascular invasion, and an unfavorable outcome in CCA. The role of sLea in vascular invasion which may lead to poor prognosis is supported by the in vitro adhesion and transmigration studies.     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

Effect of Antioxidants on Endothelial Cell Reactive Oxygen Species (ROD Generation and Adhesion of Leukocytes to Endothelial Cells

Objective To investigatewhether antioxidants inhibit adhesion of leukocytes to endothelium and furthermore, whether all antioxidants regulate NF-KB activation through a redox sensitive mechanism. Methods The effect of the antioxidative substances pyrrolidin dithiocarbamat (PDTC), dichloroisocumarin (DCI), chrysin and probucol on the endothelial leukocyte adhesion were examined under near physiological flow conditions. The antioxidative activity of antioxidants was measured in a DCF fluorescence assay with flow cytometry. The activation of NF-kB in endothelial cells was investigated in a gel shift assay. Results PDTC and probucol did not show an inhibitory effect to the formation of intracellular H2O2 in TNFa activated human vascular endothelial cells (HUVEC) . Chrysin showed a moderate effect. DCI showed a strong antioxidative effect. In contrast, PDTC and chrysin inhibited the adhesion of HL 60 cells to TNFa-stimulated HUVEC. DCI and probucol did not have influence on the adhesion within the area of th     

Endothelial FGFR1 deficiency induces AcS-DKP-resistant EndMT by regulating TGFβ signal pathway

正Objective To investigate the role of fibroblast growth factor receptor (FGFR) 1 in endothelial to-mesenchymal transition (EndMT) and epithelial-to-mesenchymal transition (EMT),and to find out a new strategy to study the vascular endothelial function of diabetic renal fibrosis.Methods Culture media from FRS2 knockdown HMVECs was transferred to HK-2 cells.Western blot and immunofluorescence staining were used to measure EMT markers and key moleculars of transforming growth factor (TGFβ).Re-