Transgenic mice with overexpression of human scavenger receptor A on endothelial cells.
OBJECTIVES: To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo. METHODS: Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-AI expression in transgenic mice. The activity of human SR-AI was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy. RESULTS: The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma I digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl II digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and immunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmission electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells. CONCLUSIONS: A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transgenic model for investigation of atherosclerosis and functions of human SR-A. (+info)
Effects of tetrandrine on cardiovascular electrophysiologic properties.
Tetrandrine (Tet) is one of the best characterized Ca2+ channel blocker of plant origin. It can affect cardiovascular electrophysiologic properties in following field: inhibit the contractility, +/-dt/dpmax, and automaticity of myocardium, prolong the FRP, and exert concentration-dependent negative inotropic and chronotropic effects without altering cardiac excitability. Tet directly blocks both T-type and L-type calcium current in ventricular cells and vascular smooth muscle cells, but it does not shift the I-V relationship curve of ICa. All its effects would be beneficial in the treatment of angina, arrhythmias, and other cardiovascular disorders. Tet also directly inhibits the activity of BKCa channel in endothelial cell line and also inhibits Ca2+-release-activated channels in vessel endothelial cells, which might significantly contribute to the change of endothelial cell activity. (+info)
Effect of Korea red ginseng on cerebral blood flow and superoxide production.
AIM: To investigate the effects of Korea red ginseng (KRG) on the cerebral perfusion rate in the rats and the generation of superoxide anion in the endothelial cells. METHODS: The cerebral perfusion rate was measured using laser-doppler flowmetry before and after the administration of crude saponin (CS) and saponin-free fraction (SFF) of KRG in the anesthetized rats. The superoxide generation was measured by the method based on lucigenin-enhanced chemiluminescence in the cultured endothelial cells. RESULTS: The relative cerebral perfusion rate (rCBF) was significantly increased by the intraperitoneal injection of CS (100 mg/kg) in the rats, but SFF had no effect on the rCBF. Chronic treatment with CS for 7 d significantly inhibited the decrease of forebrain cerebral blood flow induced by clamping both carotid arteries in the rats. Furthermore, CS (0.1 g/L) significantly suppressed NADPH-induced superoxide generation in the human umbilical vein endothelial cells (P <0.01). CONCLUSION: The present study demonstrated that crude saponin fraction of KRG enhanced cerebral blood flow in rats. Furthermore, crude saponin fraction of KRG abrogated the NADPH-driven superoxide generation in endothelial cells. (+info)
Enhancement of fibrinolytic activity of bovine aortic endothelial cells by ginsenoside Rb2.
AIM: The effect of ginsenoside Rb2 purified from Panax ginseng on fibrinolytic activity of bovine aortic endothelial cells (BAEC) was investigated. METHODS: Cellular plasminogen activator (PA) level of the lysates was measured by the chromogenic substrate S-2403. Fibrin underlay technique was carried out to observe fibrinolysis by growing endothelial cells in the culture medium. Cell viability was then determined by measurement of the activity of mitochondrial dehydrogenase. The ability of Rb2 of potentiating cellular PA activity was investigated by measuring the amounts of PA and PA inhibitor-1 (PAI-1) in the culture medium using zymography and reverse zymography. Changes in the expression of urokinase-type PA (uPA), uPA receptor, and PAI-1 mRNA in BAEC after treatment with Rb2 were analyzed by Northern blot. RESULTS: Rb2 enhanced cellular PA activity in a concentration-and time-dependent manner. Treatment of BAEC with Rb2 10 mg/L for 9 h resulted in a 3.5-fold increase of PA activity without a marked cytotoxic effect, as shown by LDH levels in culture. Increased PA levels caused the increase in surface plasmin levels as observed by fibrin underlay technique. Rb2 greatly or moderately increased the amount of urokinase-type PA (uPA) or its inhibitor (PAI-1), present in the culture medium, whereas saponin did not influence mRNA levels of uPA, its surface receptor, and PAI-1, suggesting that Rb2 may stimulate the secretion of uPA without enhancing its gene expression. The enhancement of PA levels by retinoic acid alone, a stimulator of PA synthesis, was potentiated by the simultaneous addition of ginsenoside Rb2 1 mg/L. Therefore, Rb2 might exert a strong synergism in the synthesis of cellular PA in BAEC. CONCLUSION: Ginsenoside Rb2 enhanced the PA activity levels in BAEC as well as the surface plasmin activity of BAEC. Rb2 may stimulate the secretion of uPA without enhancing the gene expression of uPA, uPA receptor (uPAR), and PAI-1. (+info)
Effect of matrine on cold ischemia and reperfusion injury of sinusoidal endothelial cells in rat orthotopic liver transplantation.
AIM: To study the mechanism and prevention of matrine (Mat) on cold ischemia/reperfusion injury of sinusoidal endothelial cells (SEC) in rat orthotopic liver transplantation (OLT). METHODS: One hundred and twenty-six syngeneic SD rats were randomly divided into four groups (n=18): untreated group, 40 mg/kg treated group, 80 mg/kg treated group, and pseudo-treated group. After 5 h of preservation in Ringer's (LR) solution, orthotopic implantation of the donor liver was performed. At 1, 2, and 4 h after reperfusion of the portal vein, 6 rats were killed in each group to collect the serum and the median lobe of liver for assay. RESULTS: The level of hylluronic acid (HA) and intercellular adhesion molecule-1 (ICAM-1) decreased significantly in both treated groups at different times post-transplantation, and their pathological changes of SEC were ameliorated, too. CONCLUSION: Matrine can prevent SEC from ischemia and reperfusion injury in rat orthotopic liver transplantation. (+info)
Endothelial cell proliferation in male reproductive organs of adult rat is high and regulated by testicular factors.
Endothelial cells in the intact adult are, apart from those in the female reproductive organs, believed to be quiescent. Systematic examination of endothelial cell proliferation in male reproductive organs has not been performed and was therefore the aim of the present study. Intact adult rats were either pulse labeled or long-term labeled with bromodeoxyuridine to label proliferating cells. The roles of Leydig cells and testosterone were examined after castration or treatment with the Leydig cell toxin ethane dimethane sulfonate (EDS) and testosterone substitution. After perfusion fixation, all blood vessels remained open and were easily identified. In all male reproductive organs studied, particularly in the testis and epididymis, endothelial cell proliferation was considerably higher than in other tissues such as the liver, brain, and muscle. Proliferating endothelial cells were observed in all types of blood vessels in male reproductive organs, but other characteristics of new blood vessel formation were not seen. High endothelial cell proliferation may reflect a continuous high turnover of endothelial cells rather than classical angiogenesis. In the epididymis, the ventral and dorsolateral prostate lobes, and the seminal vesicles, endothelial cell proliferation decreased after testosterone withdrawal and increased following testosterone treatment. In the testis, endothelial cell proliferation was decreased after Leydig cell depletion but remained low after testosterone substitution. High, hormonally regulated endothelial cell proliferation is not unique to the female but is also seen in the male reproductive organs. (+info)
Chemokine receptor expression in human endometrium.
Chemokines play a role in endometrial physiology and pathology and may affect endometrial receptivity and menstrual shedding. Chemokines exert their effect by binding to their relevant receptors, the expression levels of which may modulate their action. In the present study, we examined the expression of chemokine receptors CXCR1 and CXCR2 (receptors for interleukin-8) and CCR5 (receptor for RANTES [regulated-on-activation, normal-T-cell-expressed and -secreted], macrophage inflammatory protein [MIP]-1alpha, and MIP-1beta) in human endometrium. Human endometria (n = 35) were grouped according to the menstrual cycle phase and examined by immunohistochemistry for CXCR1, CXCR2, and CCR5. In both epithelial and stromal cells, CXCR1 and CXCR2 immunoreactivity was detected. Staining was most prominent at the apical and basal aspects of epithelial cells. Intense CCR5 immunostaining was observed in epithelial and stromal compartments throughout the menstrual cycle. Epithelial and stromal staining for CXCR1 reached a peak at the midsecretory phase, during which it was significantly higher than the level of staining during the proliferative phase (P < 0.05). Immunostaining for CXCR2 and CCR5 showed no significant variation across the menstrual cycle. Expression of interleukin-8 and RANTES in endometrium, together with the presence of their receptors, suggests that autocrine and paracrine interactions involving these chemokines may participate in endometrial physiology. (+info)
Differentiation of endothelial progenitor cells from human umbilical cord blood CD 34+ cells in vitro.
AIM: To study the time course of the expression of stem cell marker and endothelial cell markers on human cord blood CD34+ cells during in vitro differentiation process of endothelial progenitor cells (EPC). METHODS: CD34+ cells were selected and enriched from human cord blood by magnetically activated cell sorting (MACS), and cultured in dishes coated with or without fibronectin (Fn). Endothelial cells were identified by staining the cells with anti Flk-1 and vWF antibodies. The percentage of AC133+ cells in adherent CD34+ cell population was analyzed by fluorescence-activated cell sorting (FACS). RESULTS: The expression of Flk-1 and vWF on adherent CD34+ cells increased during the culture time, with 27.0 % positive for Flk-1 and negative for vWF at d 3, and 100 % positive for both Flk-1 and vWF at d 7. When cells were cultured in Fn-treated dishes, the percentages of Flk-1 and vWF positive cells increased to 34 % and 47 %, respectively at d 3, and 100 % at d 7. In contrast, the percentages of AC133+ cells among the adherent cell population decreased rapidly, and similar changes occurred in cells cultured in the presence of Fn. CONCLUSION: The gradual appearance of endothelial cell markers and the disappearance of stem cell marker characterized the in vitro differentiation of endothelial progenitor cells. Fibronectin accelerated the differentiation process of EPC. (+info)