Xukai Wang

Insulin promotes vascular smooth muscle cell proliferation via microRNA-208-mediated downregulation of p21.

Objective Abnormal vascular smooth muscle cell (VSMC) proliferation is involved in the development of vascular diseases. However, the mechanisms by which insulin exerts this effect are not completely known. We hypothesize that microRNAs might be involved in insulin-induced VSMC proliferation. Methods VSMC proliferation was determined by [3H]-thymidine incorporation; microRNAs were determined by microRNA chips and real-time PCR; and p21expression was determined by immunoblotting. Results In this study, we found that insulin increased VSMC proliferation and miR-208 expression. Overexpression of miR-208 increased basal and insulin-mediated VSMC proliferation. Although a miR-208 inhibitor, by itself, had no effect on VSMC proliferation, it reduced the insulin-mediated cell proliferation. Moreover, miR-208 increased the transformation of cell cycle from G0/G1 phase to the S phase. Bioinformatics analysis found that p21, a member of the cyclin-dependent kinase (CDK)-inhibitory protein family, may be the target of miR-208. Insulin decreased p21 expression in VSMCs; transfection of miR-208 also decreased p21 protein expression. In the presence of miR-208 inhibitor, the inhibitory effect of insulin on p21 expression in VSMCs was partially blocked. The interaction between miR-208 and p21 was direct. Using a luciferase reporter with entire wild-type p21 3′UTR or a mutant p21 3′UTR in HEK293 cells, we found that miR-208 decreased but neither miR-208 mimic nor the mutant p21 3′UTR had any significant effect on the luciferase activity. Conclusion This study indicates that miRNAs, miR-208, in particular, are involved in the insulin-induced VSMC proliferation via downregulation of its potential target, p21, a key member of CDK-inhibitory protein family.

Plasma long non-coding RNA, CoroMarker, a novel biomarker for diagnosis of coronary artery disease.

Long non-coding RNAs (lncRNAs) have been reported to be involved in the pathogenesis of cardiovascular disease (CVD), but whether circulating lncRNAs can serve as a coronary artery disease (CAD), biomarker is not known. The present study screened lncRNAs by microarray analysis in the plasma from CAD patients and control individuals and found that 265 lncRNAs were differentially expressed. To find specific lncRNAs as possible CAD biomarker candidates, we used the following criteria for 174 up-regulated lncRNAs: signal intensity ≥8, fold change >2.5 and P<0.005. According to these criteria, five intergenic lncRNAs were identified. After validation by quantitative PCR (qPCR), one lncRNA was excluded from the candidate list. The remaining four lncRNAs were independently validated in another population of 20 CAD patients and 20 control individuals. Receiver operating characteristic (ROC) curve analysis showed that lncRNA AC100865.1 (referred to as CoroMarker) was the best of these lncRNAs. CoroMarker levels were also stable in plasma. The predictive value of CoroMarker was further assessed in a larger cohort with 221 CAD patients and 187 control individuals. Using a diagnostic model with Fishers criteria, taking the risk factors into account, the optimal sensitivity of CoroMarker for CAD increased from 68.29% to 78.05%, whereas the specificity decreased slightly from 91.89% to 86.49%. CoroMarker was stable in plasma because it was mainly in the extracellular vesicles (EVs), probably from monocytes. We conclude that CoroMarker is a stable, sensitive and specific biomarker for CAD.

Randomized, double-blind, placebo-controlled trial of oral enalapril in patients with neurally mediated syncope☆☆☆★★★

BACKGROUND The purpose of this study was to study the effect of enalapril on neurally mediated syncope (NMS). Several agents (except for angiotensin-converting enzyme [ACE] inhibitors) have been used to treat patients with NMS. It is unknown whether ACE inhibitors have beneficial effects on NMS. METHODS AND RESULTS Thirty subjects who had reproducible NMS induced with head-up tilt table test (HUT) were randomly assigned and divided in double-blind fashion into placebo and enalapril (an ACE inhibitor) groups. Hemodynamics and plasma catecholamine concentrations were studied. Before administration of enalapril, syncope induced by HUT was associated with vigorous hypotension and bradycardia. Plasma catecholamine concentrations were significantly elevated during NMS compared with the supine position before tilt. Oral enalapril rather than placebo produced a marked reduction in diastolic blood pressure during supine positioning before tilt. Administration of enalapril prevented HUT-induced NMS and increase of plasma catecholamine concentrations in all patients examined. Conversely, placebo had no effect in the majority of patients with NMS (12 of 15 subjects). Follow-up data showed that NMS disappeared in 14 (93%) of 15 patients treated with enalapril. CONCLUSIONS This study demonstrates that ACE inhibitors may efficiently prevent NMS, presumably through inhibition of sympathetic system activation and peripheral hypotensive effect.

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.

Sodium butyrate alleviates adipocyte inflammation by inhibiting NLRP3 pathway.

Insulin resistance (IR) is a common feature of Type II diabetes, metabolic disorders, hypertension and other vascular diseases. Recent studies showed that obesity-induced inflammation may be critical for IR. To investigate the anti-inflammatory effect of sodium butyrate (NaB) on obesity-induced inflammation, the db/db mice were intraperitoneally injected with NaB for 6 weeks. Glucose control was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT). Adipose tissue was harvested for gene expression analysis. 3T3-L1 adipocytes were treated with Tnf-α to mimic the inflammatory state and gene expression was detected by realtime PCR and Western blotting. Our results showed that NaB treatment improved glucose control in db/db mice as determined by GTT and ITT tests. Gene expression analysis showed that NaB inhibited cytokines and immunological markers including CD68, Interferon-γ and Mcp in adipose tissues in db/db mice. Moreover, NaB inhibited cytokine releasing in 3T3-L1 adipocytes treated with TNF-α. Further analysis of inflammation pathway showed that NLRP3 was activated in db/db mice, which was efficiently inhibited by NaB treatment. Our data suggest that inhibition of obesity-induced inflammation alleviates IR, and NaB might be a potential anti-inflammatory agent for obesity.

Fixed-combination of amlodipine and diuretic chronotherapy in the treatment of essential hypertension: improved blood pressure control with bedtime dosing—a multicenter, open-label randomized study

Previous studies have demonstrated that individual anti-hypertension medications have different effects when administered in the morning vs. the evening. However, the impact of administration timing on fixed combinations of anti-hypertensive medications on blood pressure control is still unknown. In the present study, we examined the administration time-dependent effects of a fixed combination of amlodipine and diuretics (amlodipine complex) on blood pressure in hypertensive subjects. Eighty patients from Chongqing City were enrolled in this study. Subjects were randomly assigned to receive a single pill (amlodipine complex, each tablet containing amlodipine 5 mg and hydrochlorothiazide 25 mg), either in the morning (0800 hours, n=40) or at bedtime (2200 hours, n=40). Blood pressure was measured by ambulatory monitoring every 20 min during the day and every 30 min at night for 24 consecutive hours before and after the 12 weeks of treatment. Following treatment, the 24-h mean systolic and diastolic blood pressures were reduced significantly in both the morning and bedtime groups. However, the morning blood pressure surge was reduced to a greater degree in the bedtime group. In addition, the nocturnal blood pressure and the 24 h mean blood pressure were lower in the bedtime group. More patients converted from having a non-dipper to dipper blood pressure in the bedtime group. These findings confirm that amlodipine complexes have different efficiencies depending on treatment time. Administration of amlodipine complexes at bedtime could optimize the anti-hypertensive effect by augmenting blood pressure-lowering effects, increasing the diurnal/nocturnal ratio of blood pressure, normalizing the blood pressure pattern and minimizing the morning blood pressure surge.

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.

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.