Changqing Yu

(-)-Epigallocatechin gallate suppresses proliferation of vascular smooth muscle cells induced by high glucose by inhibition of PKC and ERK1/2 signalings.

Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetes-related vascular complications. (-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, is able to exert antidiabetes effects in animal models. However, it is not known whether or not EGCG inhibits VSMC proliferation induced by high glucose. This study tested the hypothesis that EGCG might have an inhibitory effect on VSMC proliferation induced by high glucose. VSMC proliferation was determined by [(3)H]-thymidine incorporation and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by immunoblotting, and ERK 1/2 activity was detected by measuring the ability to phosphorylate its substrate Elk-1. Glucose increased VSMC proliferation in a concentration-dependent manner, which was reduced in the presence of EGCG. VSMC proliferation mediated by high glucose (30 mM) was involved in protein kinase C (PKC) and ERK1/2 signalings, because its effect was blocked by PKC inhibitor (PKC inhibitor 19-31) and ERK1/2 inhibitor (PD98059). Pretreatment of VSMCs with EGCG significantly inhibited the stimulatory effect of high glucose on PKC and ERK1/2 activation, followed by attenuation of its downstream transcription factor Elk-1 phosphorylation. Taken together, these results suggest that EGCG could suppress VSMC proliferation induced by high glucose by inhibition of PKC and ERK1/2 signalings in VSMCs, which indicates that EGCG might be a possible medicine to reduce vascular complications in diabetes.

D3 Dopamine Receptor Regulation of ETB Receptors in Renal Proximal Tubule Cells From WKY and SHRs

BACKGROUND The dopaminergic and endothelin systems, by regulating sodium transport in the renal proximal tubule (RPT), participate in the control of blood pressure. The D(3) and ETB receptors are expressed in RPTs, and D(3) receptor function in RPTs is impaired in spontaneously hypertensive rats (SHRs). Therefore, we tested the hypothesis that D(3) receptors can regulate ETB receptors, and that D(3) receptor regulation of ETB receptors in RPTs is impaired in SHRs. METHODS ETB receptor expression in RPT cells was measured by immunoblotting and reverse transcriptase-PCR and ETB receptor function by measuring Na(+)-K(+) ATPase activity. D(3)/ETB receptor interaction was studied by co-immunoprecipitation. RESULTS In Wistar-Kyoto (WKY) RPT cells, the D(3) receptor agonist, PD128907, increased ETB receptor protein expression, effects that were blocked by removal of calcium in the culture medium. The stimulatory effect of D(3) on ETB receptor mRNA and protein expression was also blocked by nicardipine. In contrast, in SHR RPT cells, PD128907 decreased ETB receptor expression. Basal D(3)/ETB receptor co-immunoprecipitation was three times greater in WKY than in SHRs. The absolute amount of D(3)/ETB receptor co-immunoprecipitation induced by a D(3) receptor agonist was also greater in WKY than in SHRs. Stimulation of ETB receptors decreased Na(+)-K(+) ATPase activity in WKY but not in SHR cells. Pretreatment with PD128907 augmented the inhibitory effect of BQ3020 on Na(+)-K(+) ATPase activity in WKY but not in SHR cells. CONCLUSIONS D(3) receptors regulate ETB receptors by physical receptor interaction and govern receptor expression and function. D(3) receptor regulation of ETB receptors is aberrant in RPT cells from SHRs.

Renal D3 dopamine receptor stimulation induces natriuresis by endothelin B receptor interactions.

Dopaminergic and endothelin systems participate in the control blood pressure by regulating sodium transport in the renal proximal tubule. Disruption of either the endothelin B receptor (ETB) or D(3) dopamine receptor gene in mice produces hypertension. To examine whether these two receptors interact we studied the Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats by selectively infusing reagents into the right kidney of anesthetized rats. The D(3) receptor agonist (PD128907) caused natriuresis in WKY rats which was partially blocked by the ETB receptor antagonist. In contrast, PD128907 blunted sodium excretion in the SHRs. We found using laser confocal microscopy that the ETB receptor was mainly located in the cell membrane in control WKY cells. Treatment with the D(3) receptor antagonist caused its internalization into intracellular compartments that contained the D(3) receptors. Combined use of D(3) and ETB antagonists failed to internalize ETB receptors in cells from WKY rats. In contrast in SHR cells, ETB receptors were found mainly in internal compartments under basal condition and thus were likely prevented from interacting with the agonist-stimulated, membrane-bound D(3) receptors. Our studies suggest that D(3) receptors physically interact with proximal tubule ETB receptors and that the blunted natriuretic effect of dopamine in SHRs may be explained, in part, by abnormal D(3)/ETB receptor interactions.

Inhibitory effect of D1-like and D3 dopamine receptors on norepinephrine-induced proliferation in vascular smooth muscle cells

The sympathetic nervous system plays an important role in the regulation of blood pressure. There is increasing evidence for positive and negative interactions between dopamine and adrenergic receptors; the activation of the alpha-adrenergic receptor induces vasoconstriction, whereas the activation of dopamine receptor induces vasorelaxation. We hypothesize that the D1-like receptor and/or D3 receptor also inhibit alpha1-adrenergic receptor-mediated proliferation in vascular smooth muscle cells (VSMCs). In this study, VSMC proliferation was determined by measuring [3H]thymidine incorporation, cell number, and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Norepinephrine increased VSMC number and MTT uptake, as well as [3H]thymidine incorporation via the alpha1-adrenergic receptor in aortic VSMCs from Sprague-Dawley rats. The proliferative effects of norepinephrine were attenuated by the activation of D1-like receptors or D3 receptors, although a D1-like receptor agonist, fenoldopam, and a D3 receptor agonist, PD-128907, by themselves, at low concentrations, had no effect on VSMC proliferation. Simultaneous stimulation of both D1-like and D3 receptors had an additive inhibitory effect. The inhibitory effect of D3 receptor was via protein kinase A, whereas the D1-like receptor effect was via protein kinase C-zeta. The interaction between alpha1-adrenergic and dopamine receptors, especially D1-like and D3 receptors in VSMCs, could be involved in the pathogenesis of hypertension.

Insulin Increases D5 Dopamine Receptor Expression and Function in Renal Proximal Tubule Cells From Wistar-Kyoto Rats

BACKGROUND Ion transport in the renal proximal tubule (RPT) is regulated by numerous hormones and humoral factors, including insulin and dopamine. Previous studies show an interaction between insulin and the D(1) receptor. Because both D(1) and D(5) receptors belong to the D(1)-like receptor subfamily, it is possible that an interaction between insulin and the D(5) dopamine receptor exists in RPT cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). METHODS D(5) receptor expression in immortalized RPT cells from WKY and SHRs was quantified by immunoblotting and D(5) receptor function by measuring Na(+)-K(+) ATPase activity. RESULTS Insulin increased the expression of the D(5) receptor. Stimulation with insulin (10(-7) mol/l) for 24 h increased D(5) receptor expression in RPT cells from WKY rats. This effect of insulin on D(5) receptor expression was aberrant in RPT cells from SHRs. The stimulatory effect of insulin on D(5) receptor expression in RPT cells from WKY rats was inhibited by a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19-31, 10(-6) mol/l) or a phosphatidylinositol 3 (PI3) kinase inhibitor (wortmannin, 10(-6) mol/l), indicating that both PKC and PI3 kinase were involved in the signaling pathway. Stimulation of the D(5) receptor heterologously expressed in HEK293 cells with fenoldopam (10(-7) mol/l/15 min) inhibited Na(+)-K(+) ATPase activity, whereas pretreatment with insulin (10(-7) mol/l/24 h) increased the D(5) receptor-mediated inhibition. CONCLUSIONS Insulin and D(5) receptors interact to regulate renal sodium transport; an aberrant interaction between insulin and D(5) receptor may participate in the pathogenesis of hypertension.

Inhibitory Effect of the D3 Dopamine Receptor on Insulin Receptor Expression and Function in Vascular Smooth Muscle Cells

BACKGROUND Vascular smooth muscle cell (VSMC) proliferation is regulated by numerous hormones and humoral factors. Our previous study found that stimulation of D(1)-like dopamine receptors inhibited insulin receptor expression and function in VSMCs. We hypothesize that there is also an interaction between D(3) dopamine and insulin receptors, i.e., stimulation of the D(3) receptor inhibits insulin receptor expression and function. METHODS Receptor expression was determined by immunoblotting, immunohistochemisty, and reverse transcriptase-PCR; VSMC proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay and cell number. RESULTS Insulin receptor protein is increased in the aorta of D(3) receptor deficient mice. Stimulation of the D(3) receptor inhibited insulin receptor mRNA and protein expression and insulin-mediated VSMC proliferation, and increased protein kinase A (PKA) activity, insulin receptor phosphorylation, and degradation in immortalized aortic VSMCs (A10 cells). These effects were blocked by a PKA inhibitor, indicating that the D(3) receptor-mediated decrease in insulin receptor expression was related to a decrease in transcription/post-transcription and increased degradation, involving PKA signaling. CONCLUSIONS D(3) receptor stimulation may be a target to reduce the adverse effect of insulin in hypertension by inhibition of insulin receptor expression and function in arterial VSMCs.

D1-like receptors inhibit insulin-induced vascular smooth muscle cell proliferation via down-regulation of insulin receptor expression.

Objective Vascular smooth muscle cell (VSMC) proliferation is central to the development of vascular diseases, including hypertension, which is regulated by numerous hormones and humoral factors. Our previous study showed that the stimulatory effect of norepinephrine on VSMC proliferation is inhibited by D1-like receptors and the D3 dopamine receptor, a member of the D2-like receptor family. Insulin is a proliferative hormone but it is not known if there is any interaction between insulin and D1-like receptors. We hypothesized that Dl-like receptors may have an inhibitory effect on the insulin-induced VSMC proliferation; aberrant insulin and Dl-like receptor functions could be involved in the pathogenesis of essential hypertension. Methods VSMC proliferation was determined by [3H]-thymidine incorporation; insulin receptor mRNA and protein expressions were determined by RT-PCR, immunoblotting, and immunohistochemistry. Results Insulin increased VSMC proliferation in immortalized aortic A10 cells, determined by [3H]-thymidine incorporation. Although the D1-like receptor, by itself, had no effect on VSMC proliferation, stimulation with fenoldopam, a D1-like receptor agonist, inhibited the stimulatory effect of insulin. The inhibitory effect of fenoldopam on insulin-mediated VSMC proliferation was receptor specific, because its effect could be blocked by SCH23390, a D1-like receptor antagonist. Fenoldopam also inhibited insulin receptor mRNA and protein expression, which was time dependent and concentration dependent. A PKC or MAP kinase inhibitor blocked the inhibitory effect of fenoldopam on insulin receptor expression, indicating that PKC and MAP kinase were involved in the signaling pathway. Conclusion The inhibitory effect of D1-like receptors on insulin-mediated VSMC proliferation may play an important role in the regulation of blood pressure.

Activation of the D4 dopamine receptor attenuates proliferation and migration of vascular smooth muscle cells through downregulation of AT1a receptor expression.

Angiotensin (Ang) II has an important role in the vascular smooth muscle cell (VSMC) proliferation and migration and subsequently in the development of vascular diseases, whereas dopamine has the opposite effect. Previous studies have shown an interaction between dopamine and AT1 receptors in the kidney. The dopamine D4 receptor is expressed in arteries and has an inhibitory effect on VSMC proliferation. We hypothesized that the D4 receptor, through its interaction with the AT1a receptor, may have an inhibitory effect on Ang II-mediated VSMC proliferation and migration, which could have a pivotal role in hypertension-induced vascular remodeling. In the current study, we found that Ang II markedly induced the proliferation and migration of A10 cells, which was inhibited by the D4 receptor agonist PD168077. The activation of the D4 receptor by PD168077 inhibited AT1a receptor expression in a concentration- and time-dependent manner. These effects were attenuated by silencing the D4 receptor with a D4 receptor-targeting small interfering RNA. The D4 receptor-mediated inhibition of AT1 receptor function involved protein kinase A (PKA). The activation of the D4 receptor by PD168077 increased PKA activity in A10 cells, and the presence of a PKA inhibitor (PKA inhibitor 14–22, 10−7 mol l−1 per 24 h) blocked the inhibitory effect of the D4 receptor on AT1 receptor expression and function. The inhibitory effect of the D4 receptor on AT1 receptor expression and function was preserved in VSMCs (primary culture) from spontaneously hypertensive rats relative to VSMCs from Wistar-Kyoto rats. In conclusion, our data provide insight into the regulatory role of the D4 receptor on AT1a receptor expression and function in VSMCs and suggest that targeting the action of the D4 receptor may represent an effective therapeutic approach for the treatment of cardiovascular diseases.

CYP4F2 genetic polymorphisms are associated with coronary heart disease in a Chinese population

BackgroundTo explore the relationship between CYP4F2 gene polymorphism and coronary heart disease (CHD) in a Chinese Han population.MethodsWe selected 440 CHD patients and 440 control subjects to perform a case - control study. Four SNPs (rs2108622, rs3093100, rs3093105 and rs3093135) in CYP4F2 gene were genotyped using polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP) methods. The genotype and haplotype distributions were compared between the case and the control group.ResultsWe found both rs2108622 and rs3093105 in CYP4F2 gene were associated with the risk for CHD (P <0.01). Haplotype analysis indicated that GGGT haplotype consisted by rs2108622-rs3093100-rs3093105-rs3093135 was associated with CHD risk (OR = 4.367, 95% CI: 2.241 ~ 8.510; P < 0.001), but GGTA haplotype was associated with decreased risk for CHD (OR = 0.450, 95% CI: 0.111 ~ 0.777; P <0.001).ConclusionCYP4F2 gene polymorphisms were associated with the risk of CHD in Chinese population.

Identification of the differentially expressed genes associated with familial combined hyperlipidemia using bioinformatics analysis

The aim of the present study was to screen the differentially expressed genes (DEGs) associated with familial combined hyperlipidemia (FCHL) and examine the changing patterns. The transcription profile of GSE18965 was obtained from the NCBI Gene Expression Omnibus database, including 12 FCHL samples and 12 control specimens. The DEGs were identified using a linear models for microarray data package in the R programming language. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed. Protein-protein interaction (PPI) networks of the DEGs were constructed using the EnrichNet online tool. In addition, cluster analysis of the genes in networks was performed using ClusterONE. A total of 879 DEGs were screened, including 394 upregulated and 485 downregulated genes. Enrichment analysis identified four important KEGG pathways associated with FCHL: One carbon pool by folate, α-linolenic acid metabolism, asthma and the glycosphingolipid biosynthesis-globo series. GO annotation identified 12 enriched biological processes, including one associated with hematopoiesis and four associated with bone cell differentiation. This identification was in accordance with clinical data and experiments into hyperlipidemia and bone lesions. Based on PPI networks, these DEGs had a close association with immune responses, hormone responses and cytokine-cytokine receptors. In conclusion, these DEGs may be used as specific therapeutic molecular targets in the treatment of FCHL. The present findings may provide the basis for understanding the pathogenesis of FCHL in future studies. However, further experiments are required to confirm these results.