Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Increase in proliferation of adventitial fibroblasts at early stage of atherosclerosis in apoE(-/-) mice

AIM: To study the relationship between the proliferation of adventitial fibroblasts and the early formation of atherosclerotic lesion. METHODS: ApoE(-/-) mice and wild-type C57BL/6 black mice (6-week old) were used in this experiment. 5-Bromo-2-deoxyuridine (BrdU) was administered 24 h before the animals were sacrificed at time points of 0, 2, 4 or 10 weeks after hyperlipidic diet, respectively. The ascending aorta was removed for serial sectioning. The morphological changes of the aortic tissues were observed under microscope with HE staining. Some sections of the aorta were used to observe the BrdU labeling in the adventitia and intima at different time points by the method of immunohistochemistry. In addition the arterial adventitial fibroblasts derived from apoE(-/-) mice and C57BL/6 mice after hyperlipidic diet for 2 weeks were cultured. The proliferation of the cells was examined by BrdU incorporation. The cell cycle was examined by flow cytometry. RESULTS: In vivo the BrdU-labeled cells were first found in the adventitia of apoE(-/-) mice after feeding with hyperlipidic diet for 2 weeks, the time point that no visible intimal lesion formation was found, and then subsequently emerged in the intimal lesion. No BrdU labeling was observed in C57BL/6 mice at any time point. The number of BrdU labeled cells in the adventitial fibroblasts of apoE(-/-) mice was significantly higher than that in C57BL/6 mice in vitro (P<0.01). The adventitial fibroblasts of apoE(-/-) mice exhibited higher percentages of S and G₂/M phases than those in C57BL/6 mice (P<0.05). CONCLUSION: The proliferation of arterial adventitial fibroblasts might participate in the early formation of atherosclerotic lesions.     

Yangxue qingnao-containing serum inhibits rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

AIM: To observe the effects of Yangxue qingnao-containing serum on rat vascular smooth muscle cell (VSMC) proliferation induced by lysophosphatidic acid (LPA). METHODS: The [³H]-TdR incorporation and mitogen-activated protein kinasc (MAPK) activity were measured in cultured VSMC. End product of lipid peroxidation-MDA levels were also detected. RESULTS: 1×10^－9,1×10^－8 and 1×10^－7 mol/L LPA enhanced the cultured VSMC [³H]-TdR incorporation, increased MAPK activity and MDA content in a concentration-dependent manner. 5％, 10％ and 15％ Yangxue qingnao-containing serum concentration-dependently inhibited the increase in VSMC [³H]-TdR incorporation, MAPK activity and MDA content induced by LPA. CONCLUSIONS: LPA has a stimulating effect on VSMC proliferation. The LPA-induced intracellular signal transduction may be related to MAPK activity. Yangxue-qingnao can efficiently inhibit LPA -induced VSMC proliferation,MAPK activity and lipid peroxidation.     

Lysophosphatidic acid (LPA) induces the proliferation of astrocytes in rat via pathway of protein kinase C-α and calcium ion in vitro

AIM: To determine if lysophosphatidic acid (LPA) regulates the proliferation of astrocytes (AS) and to approach the mechanism of the process.METHODS: The cerebral AS of the neonatal SD rats were cultured in vitro and divided randomly into control group, PKC excitomotor (PMA) group, LPA group, PKC-α inhibitor (Ro31-8220) group, Ro31-8220+PMA group and Ro31-8220+LPA group. The proliferation of the cells was detected by MTT assay and flow cytometry (FCM). The concentration of intra-cellular calcium ion of the cells (［Ca2＋］i) which were labeled with Fura-2/AM was determined by ultraviolet spectrophotometer. The change of PKC-α inside the cells was observed by Western blotting.RESULTS: LPA and PMA stimulated the proliferation of AS, they also enhanced the expression of PKC-α and increased the concentration of ［Ca2＋］i. After pretreated with Ro31-8220, the abilities of LPA that mentioned above were decreased. The change of ［Ca2＋］i was associated with the diversity of PKC-α.CONCLUSION: LPA promotes the proliferation of AS via the way of PKC-α and Ca2＋.     

Relationship between the tissue neutral endopeptidase and the adrenomedullin in blood and tissues of rats with septic shock

AIM: The activity and expression of neutral endopeptidase (NEP) and the adrenomedullin (ADM) contents in various tissues were observed in septic shock and control rats to study the possible role of NEP in the change of ADM contents in tissues during septic shock. METHODS: The septic shock model of rats were established by cecal ligation and puncture (CLP). ADM contents, NEP activities, level of NEP mRNA and NEP protein were measured. RESULTS: (1) In early septic shock (ES), the ADM contents were generally higher in detected tissues, the NEP activity in left ventricle and small intestine were lower and was higher in blood than those in controls, and in lung, kidney and aorta were similar with the controls. NEP immunoreactive staining were less in lung, left ventricle, endothelium and media of aorta, but more in adventitia of aorta and kidney than those of the controls; (2) In late septic shock (LS), the ADM contents in small intestine was less but in plasma and other tissues were higher, and the NEP activity were less in plasma and other tissues than those in ES. The NEP immunoreactive staining were less in heart, endothelium and media of aorta, lung and kidney than those in ES, and was no significant change in adventitia of aorta compared with those of ES. RT-PCR found that NEP gene expression were significantly less in left ventricle, aortas, lung and small intestine than those in the controls. CONCLUSIONS: In septic shock rats, the NEP activity changes heterogeneously but the ADM contents elevate in most tissues. These results indicate that during the septic shock, the local concentrations and actions of ADM in various tissues may be regulated differently by the NEP.     

Effects of adventitial inflammation of coronary artery on the formation of atherosclerotic lesions

AIM: To study the pathogenesis of atherosclerotic lesions which were closely related to the adventitial inflammation of coronary artery (CA) in apolipoprotein E gene knockout (apoE-/-) mice. METHODS: The hearts of apoE-/- mice were cut consecutively. Three kinds of CA samples (① Infiltration of inflammatory cells at CA adventitia, without lesion; ② Infiltration of inflammatory cells at CA adventitia, with the top of extending lesion directly from aorta; ③ Infiltration of inflammatory cells at CA adventitia, with mature lesion) were chosen to represent the three stages of atherosclerotic lesion formation. HE staining, Movat staining, immunohistochemical staining and electron transmission microscopy were used respectively to identify the types of the inflammatory cells infiltrated at adventitia of coronary artery. RESULTS: The constituent ratio of macrophages which infiltrated in the CA adventitia without atherosclerotic lesions, of neutrophils which were involved in the CA adventitia with young atherosclerotic lesions and of lymphocytes in the CA adventitia with mature lesions, were 60.00%, 57.65% and 66.67%, which were higher than those in the other two groups, respectively (P<0.01). CONCLUSION: It was showed that CA adventitial inflammation might be an early event inducing the formation of atherosclerotic lesions. The CA adventitia undergoes a process from acute to chronic inflammation during the formation of atherosclerotic lesions.     

Changes in L-arginine/nitric oxide pathway of the aorta in septic shock rats

AIM:To observe the change of nitric oxide (NO) generation system in the vascular adventitia, media and intima in septic shock rats.METHODS:The septic shock model was made in rats by caecal ligation and puncture. The intima, media and adventitia of the rat aorta were separated. NO production (NO₂^-), nitric oxide synthase(NOS) activity and L-arginine (L-Arg) transport were measured, separately. Inducible NOS (iNOS) distribution was detected by immunohistochemistry.RESULTS:Both in early and late stage of septic shock, NO₂^- from the intima was decreased by 66.1% and 78.9%(P<0.01), while NO₂^- from the media was increased by 1.1 and 2.2 folds(P＜0.01), and the adventitia 9.6 and 18.6-fold (P＜0.01), as compared with the sham group, respectively. The changes of NOS activity and the L-arginine transport in the intima, the media layer and the adventitia of the aorta in the septic shock rat paralleled with that of NO₂^- in these tissues. The results of iNOS immunohistochemistry showed that there were obviously positive staining in the media layer and adventitia, especially the adventitia of the rat aortas in septic shock, as compared with that in the sham control.CONCLUSIONS:During septic shock, NO production in the aortic intima was progressively suppressed. However, it was progressively increased in the aortic medial layer and adventitia, especially the adventitia with shock processes. These changes result from different changes of L-arginine transport, NOS activity and its expression in three layers of the aorta from the septic shock rat.     

Intervention of resveatrol on activated nuclear factor-κB and expression of monocyte chemotactic protein-1 in cultured rabbit aortic smooth muscle cells induced by xanthine and xanthine oxidase

AIM: To investigate the role of resveatrol among red wine on the proliferation, activity of NF-κB and the expression of monocyte chemotactic protein-1 (MCP-1) induced by xanthine and xanthine oxidase in cultured rabbit aortic smooth muscle cells (SMC). METHODS: SMC proliferation was examined by 3-4, 5-dimethylthiazol-2-yl-2, 5-diphenylte tra zoliumbromide (MTT) metabolism, activity of NF-κB, the protein and mRNA expression of MCP-1 were detected by electrophoretic mobility shift assay (EMSA), immunohistochemitry and in situ hybridyzation in cultured rabbit aortic SMC. RESULTS: 100 μmol/L-200 μmol/L resveatrol (RES), an effective composition in red wine, was confirmed to inhibit metabolism and the activity of NF-κB as well as the protein and mRNA expression of MCP-1 in rabbit aortic SMC, which were promoted by the oxygen free radicals induced by xanthine and xanthine oxidase. CONCLUSION: Resveatrol may antagonist oxygen free radicals-induced proliferation and the activity of NF-κB as well as protein and mRNA expression of MCP-1 in cultured rabbit aortic SMC, which might play an important role in preventing atherosclerosis.     

Effects of adrenomedullin 2 on proliferation of microvascular endothelial cells from the rat cerebral cortex

AIM: To investigate the effects of adrenomedullin (ADM) 2 (AM2) on proliferation of microvascular endothelial cells from the rat cerebral cortex. METHODS: Microvascular endothelial cells (MEC) were isolated from the cerebral cortex of SD rats and cultured. The cultured cells were identified using immunocytochemistry assay with antibody for factor VIII-related antigen and randomly distributed into eight experimental groups as follows: control, AM2 10-7 mol/L, 10-8 mol/L, 10-9 mol/L, ADM, ADM+AM2, 10% fetal bovine serum (FBS) stimulated, and 10% FBS＋AM2 10^-7 mol/L groups. The proliferation of MEC was detected using ［3H］-TdR incorporation assay. RESULTS: Compared with control, AM2 (10^-7-10^-9 mol/L), ADM (10^-7 mol/L), and AM2 (10^-7mol/L) co-incubated with ADM (10^-7 mol/L) had no effects on ［3H］-TdR incorporation into the MEC (P>0.05). 10% FBS induced ［3H］-TdR incorporation increased by 87.5% (vs control, P<0.05), which was abolished by co-incubated the MEC with 10^-7 mol/L AM2 (P<0.05). CONCLUSION: AM2 inhibits FBS-stimulated proliferation of MEC from the rat cerebral cortex.     

Growth characteristics and functions of rat vascular adventitia fibroblast subpopulations

AIM: To observe in vitro monoclonal culture, proliferation characteristics and vascular functions of SD rat thoracic aorta adventitial fibroblasts. METHODS: Vascular adventitial fibroblasts were individually expanded using cloning rings. Phenotypic characteristics of the cells were identified by RT-PCR. The growth ability of the cells was determined by methyl thiazolyl tetrazolium (MTT) assay. Fluorescent quantitative PCR was used to detect the mRNA expression of preproendothelin-1 (preproET-1) induced by angiotensinⅡ (AngⅡ) and treated with AngⅡ receptor antagonist losartan and PD-123319. RESULTS: Spindle cells and round cells were isolated using cloning rings.The amplified PCR products of von Willebrand factor, CD8, desmin and myosin heavy chain was not expressed in both cells. For spindle cells, the exponential phase of growth was from the 2nd day to the 4th day after passage. For round cells, the exponential phase of growth was from the 3rd day to the 5th day after passage. The concentration dependence (0~1 000 nmol/L) of AngⅡ was observed to significantly increase the expression of preproET-1 mRNA in the round cells (P<0.01), and AngⅡ had no significant effect on the mRNA expression of preproET-1 in the spindle cells. Losartan blocked the AngⅡ-induced mRNA expression of preproET-1 in the round cells, and had no significant effect on the mRNA expression of preproET-1 in the spindle cells. CONCLUSION: Rat vascular adventitial fibroblasts can be isolated using cloning rings, and 2 different phenotypic cells are obtained. AngⅡ significantly increases the mRNA expression of preproET-1 in the round cell subpopulation, suggesting that the 2 subpopulations may play different roles in vascular remodeling and reparative process.     

Effect of dexamethasone on the adrenomedullin and its gene expression in lung tissue of asthmatic rats

AIM: To study the effect of dexamethasone (DEX) on the adrenomedullin (ADM) and its gene expression in lung tissue of asthmatic rats. METHODS: 30 healthy male SD rats were randomly divided into three groups (10 for each). The asthmatic model was established by ovalbumin inhalation and injection. The mRNA expression of ADM was examined by RT-PCR and the protein expression was detected by immunohistochemical method. The airway wall thickness, the airway smooth muscle (ASM) thickness and pulmonary tissue changes were observed under light microscope. RESULTS: The expression of ADM mRNA and protein in the asthma group A were higher than those in the control group(group C) (P<0.05), indicating that the moderate expression of ADM in asthmatic rat lung tissue is compensatory. The expression was significantly higher in DEX group (group B) than that in group A (P<0.01), indicating that DEX stimulated the expression of ADM mRNA and protein in lung tissue of asthmatic rats. CONCLUSION: The remarkable expression of ADM after the therapy of dexamethasone is one of its therapeutic mechanisms.     

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.     

Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

AIM: To study the reverse effects of saikoside (SS) on the multidrug resistance (MDR) of human leukemic cell line K562/ADM and to investigate the related mechanism. METHODS: K562 cells and K562/ADM cells in the culture were treated with SS at the concentrations of 1~100 mg/L. The inhibitory rate of the cell proliferation was measured by MTT assay. Non-cytotoxic dose of SS was determined. K562/ADM cells were treated with SS at non-cytotoxic doses of 1.25, 2.5 and 5.0 mg/L with different concentrations of adriamycin (ADM,0.05~100 mg/L). The 50% inhibitory concentration (IC₅₀) and the reversal index in all groups were determined. The cell morphology was observed after treated with SS+ADM. The effects of SS on ADM accumulation in K562/ADM cells, the cell cycle profile and apoptosis were examined by flow cytometry. RESULTS: The inhibitory rates were significantly increased in a dose-dependent manner when the cells were treated with different doses of SS (1~100 mg/L). The available reversal concentration of SS was 5.0 mg/L and the reversal index was 21.5 folds for K562/ADM cells. After treated with SS+ADM, the number of tumor cells was decreased and apoptotic cells were increased in a dose-response relationship. ADM accumulation in K562/ADM cells treated with SS was significantly higher than that in control cells (P<0.05). SS may significantly enhanced the apoptosis of K562/ADM cells treated with ADM (P<0.05). K562/ADM cells treated with SS were blocked in the stage of G₀/G₁. CONCLUSION: SS has effect on proliferation inhibition and MDR reversal in K562/ADM cell line. The reversal mechanisms of SS may be due to increasing the accumulation of chemo therapeutics in the cell, inducing the cell apoptosis and arresting the cells in G₀/G₁ phase.     

The mechanism of intercellular adhesion molecule-1 expression in endothelial cells stimulated by advanced glycosylation end products

AIM: To explore the relationship between intercellular adhesion molecule-1(ICAM-1)expression in endothelial cells(EC) and advanced glycosylation end products(AGEs) stimulation. METHODS: Murine bone marrow derived ECs was stimulated by AGEs after pretreated with anti-AGEs, anti-IL-1β and N-acetylcysteine(NAC),then SOD activity and ICAM-1 concentration and adhesion rate(AR) were evaluated. RESULTS: ECs which expressed ICAM-1 induced by AGEs showed lower SOD activity . The ICAM-1 expression as well as the increase of AR caused by AGEs stimulation could be suppressed by anti-AGEs(0.12±0.01) and NAC(0.11±0.05). Anti-IL-1β had no influence on these changes. CONCLUSION: AGEs could induce endothelial cells to express ICAM-1 in vitro, most probably due to the formation of free radicals. Besides, AGEs may stimulate other cells to secrete cytokines resulting in ICAM-1 expression in endothelial cells.     

Effect on the expression of proto-onogene c-mpl in megakaryocytes line-HEL by interleukin-13

AIM: To investigate the relevance of the proliferation of megakaryocytic cell line-HEL stimulated by the recombinant human interleukin-13 (IL-13) to the expression of pro-oncogene c-mpl in HEL cells. METHODS: MTT colorimetric assay and reverse transcrition polymerase chain reaction (RT-PCR) are separately used in this study to observe the effect on the proliferation of HEL cells and the expression of c-mpl mRNA in HEL cells by rhIL-13. RESULTS: RhIL-13 stimulated the proliferation of HEL cells and upregulated the expression of c-mpl mRNA in HEL cells. CONCLUSION: Our results suggest that rhIL-13 stimulated the proliferation of HEL cells and provide the evidence that its mechanism is partly because of increasing the pro-oncogene c-mpl expression in HEL cells.     

Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

Septic shock down-regulates L -arginine /NOS /NO pathway of platelet and aortic intima in rats

AIM: To investigate alteration and cross link of the aortic and platelet endogenous L -arginine/NOS/NO pathway induced by septic shock.METHODS: The septic shock model was made in rats by caecal ligation and puncture. NO^-₂/NO^-₃ production released from aortic and platelet was measured with Greiss assay. NOS activity and L-arginine transport activity were detected by isotope tracer method. RESULTS: Both in early and late stage of septic shock, NO^-₂/NO^-₃ production, NOS activity, and the L-arginine transport from the aorta intima and platelets were obviously decreased, while those of the aorta media and adventitia were obviously increased (P<0.01), but high-affinity L-arginine transport activity from the aorta intima and platelets was increased in early stage of septic shock (P>0.05 and P<0.05), as compared with the sham group, respectively. The inhibitory effects of NO^-₂/NO^-₃, NOS activity and the L-arginine transport showed a positive correlation between platelet and aortic intima (P<0.01). CONCLUSION: Septic shock down-regulates endogenous L-arginine/NOS/NO pathway in aortic intima and platelet, up-regulates L-arginine/NOS/NO pathway of aortic media and adventitia. Detection of the alteration of endogenous L-arginine/NOS/NO pathway in platelet might act as an indirect method to assess the endothelial dysfunction involving the pathogensis of septic shock.