Lining the inner surface of the walls of blood vessels, Endothelial cells (ECs) go beyond providing selective membrane to maintain the natural structure and function of vessels; they also synthesize varieties of vasoactive proteins to modify the pressure shift in the local flow field and hence they adapt the physiological activities of vessels. In this experiment, ELISA and RT-PCR technologies were adopted. We set up five different pressure loaded ECs groups,one non-activated cultured ECs group and one single shear stress loaded ECs group. Such a design was intended to demonstrate the effects of pressure shift on the expression of vasoactive protein synthesized by ECs [Endothelin-1(ET-1), endothelial Nitric Oxide Synthase (eNOS), Cyclooxygenase-2(COX-2) and Vascular Endothelial Growth Factor(VEGF)]. Our aim was to elucidate the mechanism of the pressure shift mediated dysfunction in ECs and the related dose-effect relationship. Based on these data, we suggest that ECs could modify the expression of vasoactive protein for adapting to the pressure shift in the local flow field; while in the process of--40 cmH2O induced ECs' dysfunction, the vasoactive proteins eNOS, COX-2 and VEGF play an important role in protecting ECs.
Cells, Cultured ; Cyclooxygenase 2 ; genetics ; metabolism ; Endothelial Cells ; metabolism ; physiology ; Endothelin-1 ; genetics ; metabolism ; Humans ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Pressure ; RNA, Messenger ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo study the effect of the Compound of traditional Chinese drugs and Benazepril on gene expression of renal endothelin and its receptor of experimental diabetic nephropathy(DN). To discove the mechanism of the compound of traditional Chinese drugs in treating DN.

METHODStreptozotocin DN model was built and influenced with the compound of traditional Chinese drugs and Benazepril. The changes of Upro, Glu, HbA1C and (PT-PCR) mRNA expression levels of ET-1, ETA-R of the renal cortex were tested, and thus the histopathological character of the kidney was analysed.

RESULTThe compound of traditional Chinese drugs and Benazepril have significant difference from normal saline in the improvement of Upro, Glu, HbA1C. The compound of traditional Chinese drugs have significant difference from Benazepril in the improvement of Glu, HbA1C. The mRNA expression level of ET-1, ETA-R of the renal cortex of DN model was raised. After influenced by the compound of traditional Chinese drugs and Benazepril, the over-expression level de-creased (still higher than normal control ones). The compound of traditional Chinese drugs were more effective than Benazepril to inhibit the proliferation of the stalk region and the third cells.

CONCLUSIONET takes part in the process of diabetic glo-Merulosclerosis. Both the compound of traditional Chinese drugs and Benazepril can influence the expression quantity from the level of gene transcription of ET and its receptor. The compound of traditional Chinese drugs can not only reduce urinary albumin of DN, but also improve blood glucose, glycosylated hemoglubin. And it can inhibit nonenzymatic glucosylation of protein as well as the proliferation of the stalk region and the third cells.

Animals ; Benzazepines ; pharmacology ; Diabetes Mellitus, Type 2 ; metabolism ; Diabetic Nephropathies ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelin-1 ; biosynthesis ; genetics ; Gene Expression ; Kidney Cortex ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A ; biosynthesis ; genetics

AIMTo investigate the molecular mechanisms of saponins from the rhizome of Anemarrhena asphodeloides Bunge.

METHODSOligonucleotide microarrays consisting of 87 probes representing 87 human cardiovascular disease-related genes were constructed. Effects of saponins on gene expression in human umbilical vein endothelial cells were analyzed by comparing hybridization of Cy 5-labeled cDNAs from saponins-treated human umbilical vein endothelial cells and Cy 3-labeled cDNAs from untreated human umbilical vein endothelial cells.

RESULTSThe results indicate that angiotensinogen gene, alpha 2A-adrenoceptor gene and endothelin-converting enzyme 1 gene were downregulated 2.8, 1.9 and 3.1 folds respectively after human umbilical vein endothelial cells were incubated in medium containing 80 mg.L-1 saponins.

CONCLUSIONThese results suggest that saponins may have beneficial effect on cardiovascular diseases by modulating the function of vein endothial cells and microarray can be used to investigate the biological action of extracts from traditional Chinese medicine.

Anemarrhena ; chemistry ; Angiotensinogen ; genetics ; metabolism ; Aspartic Acid Endopeptidases ; genetics ; metabolism ; Cells, Cultured ; Down-Regulation ; drug effects ; Endothelin-Converting Enzymes ; Endothelium, Vascular ; cytology ; metabolism ; Gene Expression ; drug effects ; Humans ; Metalloendopeptidases ; Oligonucleotide Array Sequence Analysis ; Plants, Medicinal ; chemistry ; Receptors, Adrenergic, alpha-2 ; genetics ; metabolism ; Rhizome ; chemistry ; Saponins ; isolation & purification ; pharmacology ; Umbilical Veins ; cytology ; metabolism

To systematically clarify the effects of apolipoprotein E (aopE) and low-density lipoprotein receptor (LDLR) gene mutant on hyperlipidemia, vascular inflammation impairment and pathogenesis of atherosclerosis (AS), total RNA was isolated from fresh aortas of young apoE/LDLR double knockout (apoE(-/-)/LDLR(-/-)) and wild type (WT) mice using TRIzol reagent. Then RNA was reversely transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Primer pairs were designed using primer design software according to the gene sequences available in GenBank. β-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD36, endothelin-1 (ET-1), toll-like receptor 2 (TLR2), monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and platelet-derived growth factor-α (PDGF-α). SYBR Green quantitative real-time RT-PCR was used to validate gene expressions identified by RT-PCR. Blood samples were taken from the retro-orbital venous plexus, and serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured by using biochemical techniques. Serum concentrations of circulating TNF-α, IL-1β and oxidized LDL (ox-LDL) were determined by ELISA. Frozen sections of aortic sinus were stained with Sudan IV to visualize intimal fatty lesions. The results showed that the relative expressions of IL-1β, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in apoE(-/-)/LDLR(-/-) mice at the age of 1 month were higher than those in age-matched WT mice (P<0.05, P<0.01), respectively. The expressions of PDGF-α and TNF-α in apoE(-/-)/LDLR(-/-) mice at the age of 2 months were up-regulated compared to those in age-matched WT mice (P<0.05). All the expressions of target genes continued to be up-regulated (P<0.05, P<0.01) except that ET-1 expression at the age of 2 months, TLR2, VCAM-1 and ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-κB expression had no significant changes between two genotype mice at different ages. All the gene expressions kept unchanged in WT mice at different ages, except that IL-1b expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-α, IL-1β and ox-LDL in apoE(-/-)/LDLR(-/-) mice at different ages were higher than those in age-matched WT mice (P<0.05, P<0.01), and were increasing with age. Primary atherosclerotic lesions were observed in 1-month old apoE(-/-)/LDLR(-/-) mice and were progressing with age. There were no lesions observed in all WT mice at different ages. The data suggest that hyperlipidemia due to apoE and LDLR gene mutant may stimulate the temporal expressions of AS-related genes and contribute to primary atherogenetic lesions and vascular inflammation impairment.
Animals ; Aorta ; metabolism ; Apolipoproteins E ; genetics ; Atherosclerosis ; genetics ; CD36 Antigens ; metabolism ; Chemokine CCL2 ; metabolism ; Endothelin-1 ; metabolism ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Hyperlipidemias ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; blood ; metabolism ; Lipoproteins, LDL ; blood ; Mice ; Mice, Knockout ; NF-kappa B ; metabolism ; Platelet-Derived Growth Factor ; Receptors, LDL ; genetics ; Toll-Like Receptor 2 ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.
Animals ; Cells, Cultured ; Chorionic Gonadotropin/*pharmacology ; Echinomycin/*pharmacology ; Endothelin-2/genetics/*metabolism ; Female ; Gonadotropins, Equine/*pharmacology ; Granulosa Cells/drug effects/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*antagonists & inhibitors/genetics/metabolism/physiology ; Oligonucleotides/genetics ; Ovary/cytology/drug effects/physiology ; Rats ; Rats, Sprague-Dawley ; Superovulation/*drug effects ; Transcriptional Activation

Endothelin systems are believed to play important roles in the emergence and maintenance of functions of various organs during perinatal development, including the kidney. The present study was designed to investigate the roles of endothelin systems on physiologic renal growth and development. Newborn rat pups were treated with either Bristol-Myers Squibb-182874 (30 mg/kg/day), a selective endothelin A receptor (ET(A)R) antagonist, or vehicle for 7 days. To identify cellular changes, kidneys were examined for apoptotic cells by terminal deoxynucleotide transferasemediated nick-end labeling stain and proliferating cell nuclear antigen (PCNA) by immunohistochemical (IHC) stain. To clarify the molecular control of these processes, immunoblots and reverse transcriptase-polymerase chain reaction for Clusterin, Bcl-2, Bcl-X(L), Bax, and p53 were performed. ETAR antagonist treatment resulted in reduced kidney weights, decreased PCNA-positive proliferating cells, and increased apoptotic cells. The protein expressions of renal Bcl-X(L) and Bax in the ETAR antagonist-treated group were significantly decreased, whereas the mRNA expressions of these genes were not changed. There were no significant differences in the expressions of Clusterin, Bcl-2, and p53. In conclusion, inhibition of endogenous endothelins impairs renal growth, in which decreased cellular proliferation, increased apoptosis and decreased expressions of renal Bcl-X(L) and Bax are possibly implicated.
Animals ; Animals, Newborn ; Antihypertensive Agents/*pharmacology ; *Apoptosis ; *Cell Proliferation ; Dansyl Compounds/*pharmacology ; Gene Expression Regulation, Developmental/drug effects ; In Situ Nick-End Labeling ; Kidney/drug effects/*growth & development/*metabolism ; Proliferating Cell Nuclear Antigen/metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/*antagonists & inhibitors/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53/genetics/metabolism ; bcl-X Protein/genetics/metabolism

OBJECTIVETo study the features of vascular smooth muscle cell (VSMC) proliferation induced by endothelin-1 (ET-1).

METHODSVSMCs of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats were cultured and treated with ET-1. Basic fibroblast growth factor (bFGF) gene expression was measured using both Northern blot and an enzyme-linked immunoassay.

RESULTSET-1 resulted in an increase in bFGF transcripts at 8 - 24 h; bFGF levels were significantly higher in VSMCs treated with ET-1 than in those not treated. However, VSMCs growth responses in SHR and WKY were different. Smooth muscle cells of SHR were hyper-responsive to ET-1. Maximal bFGF mRNA levels were elevated 3.5-fold at 4 h of stimulation in WKY and 8-fold at 8h in SHR4. Moreover, the proliferation of VSMCs induced by ET-1 was inhibited by antisense phosphorothioate oligodeoxynucleotides (10 micromol/L AS-bFGF) but not sense bFGF oligomers at the same concentrations, being reduced by 80% in SHR and 40% in WKY vs control, respectively. Furthermore, the effect of AS-bFGF oligomers on SHR SMC proliferation is significantly greater than on WKY SMC proliferation.

CONCLUSIONET-1 may be required for exaggerated vascular growth responses in SHR and bFGF may be involved.

Animals ; Cell Division ; drug effects ; Cells, Cultured ; DNA, Antisense ; pharmacology ; Dose-Response Relationship, Drug ; Endothelin-1 ; pharmacology ; Fibroblast Growth Factor 2 ; genetics ; Gene Expression Regulation ; drug effects ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Oligonucleotides ; pharmacology ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Time Factors

OBJECTIVETo investigate 7 gene expression profile associated with inflammation and oxidative stress in vascular endothelium injure of rats with deficiency of vital energy or qi stagnation, and the effect of Tongxinluo on gene expression profile.

METHODThe model of vascular endothelium injury of rats with deficiency of vital energy or qi stagnation were established by using high L-methionine, with load-carrying swimming or being fastened, respectively. RT-PCR and SAGE database which is available in NCBI, were used to analyze the changes of 7 gene expression related with inflammation and oxidative stress in endothelium injure and the effect of Tongxinluo on the gene expression profile.

RESULTCompared with control group, the gene expression of inflammation related COX-1, COX-2, oxidative stress related iNOS, SOD and blood vessel vasomotion related eNOS, ECE, increased in deficiency of vital energy group (P < 0.05 or P < 0.01), and the gene expression decreased with Tongxinluo treatment (P < 0.05 or P < 0.01). The gene expression of COX-1, COX-2, iNOS and eNOS, ECE, increased (P < 0.01), but the gene expression of PCS and SOD decreased (P < 0.01), in qi stagnation group, and the disorder of gene expression improved with treatment of Tongxinluo (P < 0.01).

CONCLUSIONThe 7 gene expression related to vascular endothelium injure were not the same in rat with deficiency of vital energy or qi stagnation, and Tongxinluo could regulate the disorder of the gene expression, protecting vascular endothelium from injure.

Animals ; Aspartic Acid Endopeptidases ; genetics ; Cyclooxygenase 1 ; genetics ; Cyclooxygenase 2 ; genetics ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Endothelin-Converting Enzymes ; Endothelium, Vascular ; injuries ; physiopathology ; Gene Expression ; drug effects ; Gene Expression Profiling ; Male ; Medicine, Chinese Traditional ; Metalloendopeptidases ; genetics ; Nitric Oxide Synthase Type II ; genetics ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Qi ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Superoxide Dismutase ; genetics