De-Jian Jiang

Reduction of NO- and EDHF-Mediated Vasodilatation in Hypertension: Role of Asymmetric Dimethylarginine

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS), and endothelial dysfunction is related to the elevation of ADMA level in hypertension. Besides the NO-mediated pathway, the endothelium-derived hyperpolarizing factor (EDHF)-mediated pathway is involved in endothelial dysfunction. The aims of the present study were to evaluate the changes of endothelium-dependent dilatation of arteries in hypertension and the role of ADMA in NO- and EDHF-mediated vasodilatation. The great omental arteries were isolated from essential hypertensive and normotensive patients, and mesenteric arteries were isolated from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. NO-, EDHF-, and prostaglandin (PGI2)-mediated endothelium-dependent vasodilatation were measured, and plasma concentrations of ADMA were determined in rats. Cultured endothelial cells were treated with ADMA (1–10 μM) for 48 h, and the mRNA and protein level of small-conductance Ca2+-activated K+ channel 3 (SK3), which has been thought to be a key mediator of EDHF, was determined. Both NO- and EDHF-mediated endothelium-dependent responses were decreased in the great omental arteries of hypertensive patients and mesenteric arteries of SHR. Plasma levels of ADMA were significantly increased in SHR. In cultured endothelial cells, the expressions of SK3 mRNA and protein were concentration-dependently down-regulated in the presence of ADMA. The present study suggests that the inhibitory effect of ADMA on endothelial function not only involves NO-mediated endothelium-dependent vasodilatation but also the EDHF-mediated pathways in hypertensive animals and humans, and that ADMA can down-regulate the expression of SK3 channels in endothelial cells.

Asymmetric dimethylarginine induces TNF-α production via ROS/NF-κB dependent pathway in human monocytic cells and the inhibitory effect of reinioside C

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, has been implicated in vascular inflammation through induction of reactive oxygen species (ROS) and proinflammatory genes in endothelial cells. However, relatively few attentions have been paid to the effect of ADMA on monocytes, one of the important cells throughout all stages of atherosclerosis. In the present study, we found that reinioside C, the main component extracted from Polygala fallax Hemsl., dose-dependently inhibited tumor necrosis factor-alpha (TNF-alpha) production induced by ADMA in monocytes, Furthermore, reinioside C attenuated ADMA-induced generation of reactive oxygen species and activation of nuclear factor-kappaB (NF-kappaB) activity in monocytes in a dose-dependent manner, this effect was inhibited by l-arginine (NOS substrate) and PDTC (inhibitor of NF-kappaB). These data suggest that reinioside C could attenuate the increase of TNF-alpha induced by exogenous ADMA through inhibition ROS/NF-kappaB pathway in monocytes.

Regulation by DDAH/ADMA pathway of lipopolysaccharideinduced tissue factor expression in endothelial cells

Previous studies have shown the regulatory effect of nitric oxide (NO) on endotoxin-induced tissue factor (TF) in endothelial cells. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor, could inhibit NO production in vivo and in vitro. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endogenous NO production. The aim of the present study was to determine whether the DDAH/ADMA pathway is involved in the effect of lipopolysaccharide (LPS) on TF expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with LPS (1 microg/ml) to induce TF expression. Exogenous ADMA significantly enhanced the increase in both TF mRNA level and activity induced by LPS, whereas L-arginine, the NOS substrate, markedly attenuated the LPS-induced TF increment. LPS markedly increased the level of ADMA in cultured medium and decreased DDAH activity in endothelial cells, and over-expression of DDAH2 could significantly suppress LPS-induced TF increment in endothelial cells. LPS could increase intracellular reactive oxygen species (ROS) production and activate nuclear factor-kappaB, which were enhanced by exogenous ADMA and attenuated by either L-arginine or overexpression of DDAH2. Therefore, our present results for the first time suggest that the DDAH/ADMA pathway can regulate LPS-induced TF expression via ROS-nuclear factor-kappaB-dependent pathway in endothelial cells.

Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats.

We have previously reported that Chinese traditional medicine rutaecarpine (Rut) produced a sustained hypotensive effect in phenol-induced and two-kidney, one-clip hypertensive rats. The aims of this study are to determine whether Rut could exert antihypertensive and anti-platelet effects in spontaneously hypertensive rats (SHR) and the underlying mechanisms. In vivo, SHR were given Rut and the blood pressure was monitored. Blood was collected for the measurements of calcitonin gene-related peptide (CGRP), tissue factor (TF) concentration and activity, and platelet aggregation, and the dorsal root ganglia were saved for examining CGRP expression. In vitro, the effects of Rut and CGRP on platelet aggregation were measured, and the effect of CGRP on platelet-derived TF release was also determined. Rut exerted a sustained hypotensive effect in SHR concomitantly with the increased synthesis and release of CGRP. The treatment of Rut also showed an inhibitory effect on platelet aggregation concomitantly with the decreased TF activity and TF antigen level in plasma. Study in vitro showed an inhibitory effect of Rut on platelet aggregation in the presence of thoracic aorta, which was abolished by capsazepine or CGRP(8-37), an antagonist of vanilloid receptor or CGRP receptor. Exogenous CGRP was able to inhibit both platelet aggregation and the release of platelet-derived TF, which were abolished by CGRP(8-37). The results suggest that Rut exerts both antihypertensive and anti-platelet effects through stimulating the synthesis and release of CGRP in SHR, and CGRP-mediated anti-platelet effect is related to inhibiting the release of platelet-derived TF.

All-trans retinoic acid inhibits cobalt chloride-induced apoptosis in PC12 cells: Role of the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine pathway

Previous studies have shown that the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its specific hydrolase dimethylarginine dimethylaminohydrolase (DDAH) are involved in the regulation of apoptosis in different cell types. In the present study, we investigated the role of the DDAH/ADMA pathway in cobalt chloride (CoCl2)–induced apoptosis and the antiapoptotic effect of all‐trans retinoic acid (atRA) in undifferentiated pheochromocytoma (PC12) cells. Treatment of CoCl2 (125 μM) for 48 hr significantly induced the apoptosis of PC12 cells, concomitantly with increased intracellular reactive oxygen species (ROS) production and caspase‐3 activity. CoCl2 treatment also decreased the activity of DDAH and the expression of DDAH2 (mRNA and protein), resulting in an increased level of ADMA. All these alterations induced by CoCl2 were attenuated by atRA (0.1, 1, or 10 μM). Interestingly, the antiapoptotic effects of atRA were inhibited by DDAH2 small RNA interference. In contrast, DDAH2 overexpression inhibited the proapoptotic effects of CoCl2. We also found that treatment of exogenous ADMA (3, 10, or 30 μM) induced the apoptosis of PC12 cells in a concentration‐ and time‐dependent manner, which was inhibited by the antioxidant or the caspase‐3 inhibitor. These findings suggest that the modulation of the DDAH/ADMA/ROS pathway plays an important role in CoCl2‐induced apoptosis and the antiapoptotic effects of atRA in undifferentiated PC12 cells.

Involvement of dimethylarginine dimethylaminohydrolase‐2 in visfatin‐enhanced angiogenic function of endothelial cells

Visfatin, a new adipocytokine, was reported to promote angiogenesis. Dimethylarginine dimethylaminohydrolase (DDAH), which could regulate vascular endothelial growth factor (VEGF) expression in endothelial cells, is thought as a novel modulator of angiogenesis. The aim of the study was to investigate the role of DDAH2 in visfatin‐induced angiogenesis in human umbilical vein endothelial cells (HUVECs).

Delayed cardioprotection afforded by nitroglycerin is mediated by α-CGRP via activation of inducible nitric oxide synthase

Previous investigations have demonstrated that delayed preconditioning induced by nitroglycerin is mediated by endogenous calcitonin gene-related peptide (CGRP). In the present study, we examined whether CGRP-mediated delayed preconditioning induced by nitroglycerin is involved in activation of inducible nitric oxide synthase (iNOS). Male Wistar rats were pretreated with nitroglycerin 24 h before the experiment, and then the left main coronary artery of rat heart was subjected to 60-min occlusion followed by 3 h reperfusion. Infarct size, the plasma level of cGMP and CGRP, and expression of CGRP isoforms (alpha-CGRP and beta-CGRP) mRNA in lumbar dorsal root ganglia were measured. Pretreatment with nitroglycerin (120 microg/kg, i.v.) markedly reduced infarct size. Nitroglycerin caused a significant increase in the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA, concomitant with an increase in plasma concentrations of cGMP and CGRP. These effects of nitroglycerin were completely abolished by pretreatment with aminoguanidine (300 mg/kg, i.p.), a selective inhibitor of iNOS activity, or dexamethasone (5 mg/kg, i.p.), the iNOS expression inhibitor. The present results suggest that delayed cardioprotection afforded by nitroglycerin is mediated by the alpha-CGRP isoform via generation of NO derived from iNOS.

Asymmetric dimethylarginine induces tissue factor expression in monocytes via NF-kappaB-dependent pathway: Role in acute coronary syndromes

Asymmetric dimethylarginine (ADMA), a major endogenous inhibitor of nitric oxide synthase, is recently defined as an independent cardiovascular risk factor. Tissue factor (TF) expression and procoagulant activity (PCA) of peripheral monocytes are increased in patients with acute coronary syndromes (ACS), which resulted in blood procoagulant state tending to thrombus formation. In the present study, we tested the hypothesis that ADMA contribute to TF expression of peripheral monocytes in ACS. Twenty patients with unstable angina (UA), 20 patients with stable angina (SA) and 20 control subjects were recruited. Monocytic cell line THP-1 was incubated with different concentrations of ADMA (1-10microM) for various periods (6-24h). Our results showed that plasma level of ADMA in patients with UA was significantly higher than those in patients with SA or in the control group, and positively correlated with TF antigen level and PCA of circulating monocytes. Adjusting for all patient characteristics, we confirmed these findings in multivariate regression analyses. In cultured THP-1 cells, ADMA transcriptionally upregulated both TF antigen expression and PCA in a concentration-dependent manner. The experiments using nuclear factor-kappaB (NF-kappaB) inhibitor and transient transfection with wild-type and mutated TF promotor constructs showed that the NF-kappaB is an important transcriptional regulator of ADMA-induced TF expression. Our results suggest that elevated plasma level of ADMA induces TF expression in monocytes via NF-kappaB-dependent pathway, which contributes to procoagulant phenotype of circulating monocytes in ACS.

3,4,5,6-Tetrahydroxyxanthone protects against myocardial ischemia-reperfusion injury in rats.

In the present study, we tested the protective effect of 3,4,5,6-tetrahydroxyxanthone, a synthetic xanthone derivative, on myocardial ischemia-reperfusion injury in rats. Ischemia-reperfusion injury was induced by 30 min of global ischemia and 30 min of reperfusion in isolated rat hearts or 30 min coronary artery occlusion and 120 min reperfusion in vivo, respectively. Heart rate, coronary flow (CF), left ventricular pressure (LVP), and its first derivative (±dp/dtmax) were recorded, and the activity of creatine kinase in coronary effluent and tumor necrosis factor-alpha (TNF-α) content in myocardial tissues were measured in vitro. The activity of serum creatine kinase, the level of TNF-α and interleukin-6 (IL-6), and myocardial infarct size were measured in vivo. 3,4,5,6-tetrahydroxyxanthone (30, 100 or 300 μM) caused a significant improvement of cardiac function (LVP and ±dp/dtmax) and a decrease in the release of creatine kinase in coronary effluent as well as the level of TNF-α in myocardial tissues in vitro. 3,4,5,6-tetrahydroxyxanthone (0.5 or 1.0 mg/kg, i.v.) also markedly decreased infarct size and the release of creatine kinase and TNF-α, and increased serum IL-6 level in vivo. These results suggest that 3,4,5,6-tetrahydroxyxanthone possesses a protective effect on myocardial ischemia-reperfusion injury, and that the protective effects of 3,4,5,6-tetrahydroxyxanthone may be related to inhibition of TNF-α production and stimulation of IL-6 generation by inhibition of ROS production.

Rutaecarpine inhibits hypoxia/reoxygenation-induced apoptosis in rat hippocampal neurons.

Our previous studies showed that rutaecarpine (Rut) protected against myocardial ischemia/reperfusion (I/R) injury, which was associated with activation of transient receptor potential vanilloid subtype 1 (TRPV1). Recently, TRPV1 activation was also reported to exert neuroprotective effects. The present study was to investigate the effect of Rut on hypoxia/reoxygenation (H/R)-induced apoptosis in primary rat hippocampal neurons. Three-hour hypoxia (1% O2) and consequent 24-h reoxygenation significantly increased the apoptotic death of hippocampal neurons as evidenced by increases in both TUNEL-positive cell number and caspase-3 activity. However, pretreatment with Rut (1-10microM) or caspase-3 specific inhibitor DEVD-CHO could markedly attenuate H/R-induced apoptosis in neurons. Rut markedly induced the phosphorylation of Akt and PI3K inhibitor LY294002 prevented the survival effect of Rut on neurons. Intracellular oxidative stress was significantly induced after H/R, which was inhibited by Rut and LY294002 as well as antioxidant PDTC. TRPV1 antagonist capsazepine or intracellular Ca2+ chelator BAPTA/AM could abolish these effects of Rut mentioned above. In summary, the present data suggest that Rut inhibits H/R-induced apoptosis of hippocampal neurons via TRPV1-[Ca2+]i-dependent and PI3K/Akt signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.