Asymmetric dimethylarginine aggravates blood-retinal barrier breakdown of diabetic retinopathy via inhibition of intercellular communication in retinal pericytes.

Blood-retinal barrier breakdown is the main pathological characteristics of diabetic retinopathy (DR). Asymmetric dimethylarginine (ADMA) was reported to be elevated in DR patients. In this study, we observed the dynamic profile of ADMA, retinal morphology and permeability of BRB at 2, 4 or 8 week of diabetic rats induced by a single intraperitoneal injection of streptozocin (60 mg/kg) and in cultured rat retinal pericytes pretreated with D-glucose (30 mM) for 1, 3, 5 and 7 days or ADMA (3, 10, 30 µM) for 24, 48 and 72 h, trying to explore the effects of ADMA on blood-retinal barrier in DR. Gap junction intercellular communication (GJIC) and the expression of blood-retinal barrier-specific component connexin 43 (Cx43) were examined in diabetic rats or cultured retinal pericytes to elucidate whether ADMA impacted blood-retinal barrier function via damaging Cx43-GJIC. The results showed that with increasing duration of diabetes, the ultrastructure of blood-retinal barrier of diabetic rats appeared cell junction damage, apoptosis of retinal pericytes and breakdown of barrier successively. The increases in retinal permeability, ADMA levels and Cx43 expression, and abnormal GJIC were observed in diabetic rats and retinal pericytes exposed to D-glucose (30 mM). A glucose-like effect was seen using ADMA or another L-arginine analogue NG-monomethyl-L-arginine or dimethylarginine dimethylaminohydrolases (DDAHs) siRNA, implicating that ADMA aggravated the breakdown of blood-retinal barrier via damaging Cx43-GJIC.

Involvement of epithelial-mesenchymal transition afforded by activation of LOX-1/ TGF-ß1/KLF6 signaling pathway in diabetic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Diabetic pulmonary fibrosis is a severe disease that increases mortality risk of diabetes. However, the molecular mechanisms leading to pulmonary fibrosis in diabetes are poorly understood. This study investigated the roles of epithelial-mesenchymal transition (EMT) and the associated molecular mechanisms in streptozotocin (STZ)-induced rat pulmonary fibrosis. METHODS: The rat model of diabetic pulmonary fibrosis was established by intraperitoneal injection of a single dose of STZ (35 mg/kg). Typical lesions of diabetic pulmonary fibrosis were observed 8 weeks after STZ injection by hematoxylin-eosin (HE) and Masson staining. Human bronchial epithelial cells (HBECs) and A549 cells were treated by high glucose. Gene or protein expression was measured by real-time PCR, Western blot, immunohistochemistry or immunofluorescence. The knockdown of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) or transforming growth factor-ß1 (TGF-ß1) was conducted by siRNA. RESULTS: Activation of EMT was observed in lung tissues of STZ-induced diabetic rats, exhibiting a loss in the epithelial cell marker E-cadherin and an increase in the mesenchymal marker Vimentin. The protein and mRNA levels of LOX-1, TGF-ß1 and krüppel-like factor 6 (KLF6) in the lung tissues were increased. Incubation of HBECs and A549 cells with high glucose activated EMT and induced an increase in LOX-1, TGF-ß1 and KLF-6 expression. LOX-1 siRNA inhibited high glucose-induced EMT in HBECs and A549 cells, which correlated with the reduction of TGF-ß1. TGF-ß1 siRNA decreased the expression of LOX-1 and KLF6. CONCLUSIONS: EMT was involved in the pathological process of diabetic pulmonary fibrosis, which was activated by LOX-1/TGF-ß1/KLF6 signaling pathway.

Role of Asymmetrical Dimethylarginine in Diabetic Microvascular Complications.

Microvascular complications are the leading causes of acquired blindness, end-stage renal failure, and varieties of neuropathy associated with diabetes. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is involved in endothelial dysfunction, oxidative stress, and inflammation associated with the progression of diabetic microvascular complications. Elevated ADMA has been detected in experimental animals and patients with diabetic microangiopathy like retinopathy, nephropathy, and neuropathy. In the review, we focus on the role of ADMA in the pathobiology of major microvascular complications of diabetes.

Accelerated onset of senescence of endothelial progenitor cells in patients with type 2 diabetes mellitus: role of dimethylarginine dimethylaminohydrolase 2 and asymmetric dimethylarginine.

The risk of cardiovascular complications in diabetic patients is mainly associated with endothelial dysfunction. Reduced number of EPCs and impaired function of EPCs in diabetes result in imbalance of endothelial homeostasis and dysfunction of vessels. In patients with diabetes mellitus, plasma levels of asymmetric dimethylarginine (ADMA) were elevated, while the expression and activity of dimethylarginine dimethylaminohydrolase (DDAH) were reduced. In the present study, we investigated the role of the DDAH2/ADMA pathway in the senescence of EPCs in type 2 diabetic patients and cultured EPCs treated with high glucose. The results showed that the percentage of senescent EPCs increased while the expression of DDAH2 decreased concomitantly with an increase in the plasma levels of ADMA in patients with type 2 diabetes mellitus (T2DM). Similar results were seen in cultured EPCs treated with high glucose. Exogenous application of ADMA accelerated the senescence of EPCs in a dose-dependent manner, and overexpression of DDAH2 inhibited high glucose-induced EPCs senescence. In addition, it has also been reported that DDAH/ADMA pathway is regulated by silent information regulator 1 (SIRT1) in endothelial cell. In the present study, we found decreased expression of SIRT1 both in T2DM patients and EPCs pretreated with high glucose. And resveratrol (activating SIRT1) inhibited high glucose-induced EPCs senescence by upregulating the expression of DDAH2 and decreasing the levels of ADMA. Taken together, we concluded that DDAH2/ADMA is involved in the accelerated senescence of EPCs in diabetes, which is associated with the activation of SIRT1.

Quantitative phosphoproteomics analysis reveals broad regulatory role of heparan sulfate on endothelial signaling.

Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.

Dimethylarginine dimethylaminohydrolase 1 regulates nerve growth factor-promoted differentiation of PC12 cells in a nitric oxide-dependent but asymmetric dimethylargenine-independent manner.

There are significant morphological and biochemical alterations during nerve growth factor (NGF)-promoted neuronal differentiation, and the process is regulated by molecules, including nitric oxide (NO). Dimethylarginine dimethylaminohydrolase (DDAH) is thought to play a critical role in regulating NO production via hydrolyzing the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus, we tested the role of DDAH in NGF-promoted differentiation of PC12 (pheochromocytoma) cells. The present results show that both mRNA and protein levels of DDAH1 were increased, whereas those of DDAH2 were decreased, during NGF-promoted cell differentiation. Both the DDAH activity and the ADMA level in cultured medium were unchanged in this process. NGF promoted neurite formation and induced the expression of microtubule-associated protein 2 (MAP2), a neuronal marker, which were both significantly repressed by DDAH1 silence with small interfering RNA but not by DDAH2 silence. The expressions of three isoforms of NOS were markedly upregulated after NGF stimulation with a time course similar to that of DDAH1, which were attenuated by DDAH1 silence. Conversely, overexpression of DDAH1 accelerated neurite formation in PC12 cells, concomitantly with upregulating the expression of three NOS isoforms. In summary, our data reveal the critical regulatory effect of DDAH1 on NGF-promoted differentiation of PC12 cells in an NOS/NO-dependent but ADMA-independent manner.

Involvement of miR-199a-3p/DDR1 in vascular endothelial cell senescence in diabetes.

Endothelial cell dysfunction is a prominent feature of diabetic cardiovascular complications, and endothelial cell senescence is considered to be an important contributor to endothelial dysfunction. Discoidin domain receptor 1 (DDR1) has been reported to be involved in atherogenesis and cerebral ischemia/reperfusion injury. In this study, we aimed to explore the role of DDR1 in endothelial cell senescence under diabetic conditions and elucidate the underlying mechanisms. A diabetic rat model was established by a single intraperitoneal injection of streptozocin (STZ) (60 mg/kg), which showed an increase in senescence-associated ß-galactosidase (SA-ß-gal) staining signal of thoracic aortic endothelium, impaired vascular structure and function, accompanied by an up-regulation of DDR1. Next, we verified the role of DDR1 in endothelial senescence and the underlying mechanisms in high glucose-treated human umbilical vein endothelial cells (HUVECs). Consistent with the in vivo findings, high glucose induced endothelial senescence, impaired endothelial function and elevated DDR1 expression, accompanied by the elevation of senescence-related genes p53 and p21 expression, and these effects were reversed by DDR1 siRNA. DDR1 has been documented to be a potential target of miR-199a-3p. Here, we found that miR-199a-3p was down-regulated by high glucose in the aorta tissue and HUVECs, while miR-199a-3p mimic significantly suppressed increased endothelial senescence and elevated DDR1 induced by high glucose. In conclusion, our data demonstrated that miR-199a-3p/DDR1/p53/p21 signaling pathway was involved in endothelial senescence under diabetic conditions, and therapeutic targeting DDR1 would be exploited to inhibit endothelial senescence owing to high glucose exposure.

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 (CoCl(2))-induced apoptosis and the antiapoptotic effect of all-trans retinoic acid (atRA) in undifferentiated pheochromocytoma (PC12) cells. Treatment of CoCl(2) (125 microM) for 48 hr significantly induced the apoptosis of PC12 cells, concomitantly with increased intracellular reactive oxygen species (ROS) production and caspase-3 activity. CoCl(2) 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 CoCl(2) were attenuated by atRA (0.1, 1, or 10 microM). Interestingly, the antiapoptotic effects of atRA were inhibited by DDAH2 small RNA interference. In contrast, DDAH2 overexpression inhibited the proapoptotic effects of CoCl(2). We also found that treatment of exogenous ADMA (3, 10, or 30 microM) 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 CoCl(2)-induced apoptosis and the antiapoptotic effects of atRA in undifferentiated PC12 cells.

Asymmetric dimethylarginine: An crucial regulator in tissue fibrosis.

Fibrosis is a reparative process with very few therapeutic options to prevent its progression to organ dysfunction. Chronic fibrotic diseases contribute to an estimated 45% of all death in the industrialized world. Asymmetric dimethylarginine (ADMA), an endothelial nitric oxide synthase inhibitor, plays a crucial role in the pathogenesis of various cardiovascular diseases associated with endothelial dysfunction. Recent reports have focused on ADMA in the pathogenesis of tissue fibrosis. This review discusses the current knowledge about ADMA biology, its association with risk factors of established fibrotic diseases and the potential pathophysiological mechanisms implicating ADMA in the process of tissue fibrosis.

Asymmetric dimethylarginine contributes to retinal neovascularization of diabetic retinopathy through EphrinB2 pathway.

Diabetic retinopathy (DR) is a leading cause of vision loss with retinal neovascularization. This study aims to investigate whether Asymmetric dimethylarginine (ADMA) impacts the pathogenesis of DR via focusing on promoting retinal neovascularization and its underlying molecular mechanisms. Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ) for 20 weeks. ADMA levels in aqueous and the influence of hypoxia on ADMA and angiogenesis in RF/6A cells were examined. The effects and underlying molecular mechanisms of ADMA on neovascularization of RF/6A cells were further evaluated by administration of ADMA, DDAH siRNA or ephrinB2 siRNA. Results showed that ADMA levels were elevated in both aqueous from diabetic rats and culture medium in RF/6A cells pretreated with hypoxia. Administration of ADMA directly promoted proliferation, migration, adhesion and tube formation of RF/6A cells, which was further confirmed by DDAH1 siRNA or DDAH2 siRNA. In addition, ephrinB2 expression was increased under diabetic conditions, and the angiogenic effects of ADMA were blocked by ephrinB2 siRNA. In conclusion, ADMA contributes to the neovascularization of retina in diabetic mellitus, which is regulated by ephrinB2.

Taurine protects against low-density lipoprotein-induced endothelial dysfunction by the DDAH/ADMA pathway.

Asymmetric dimethylarginine (ADMA), a major endogenous nitric oxide (NO) synthase inhibitor, is thought to be a key contributor for endothelial dysfunction. Decrease in activity of dimethylarginine dimethylaminohydrolase (DDAH), a major hydrolase of ADMA, causes accumulation of ADMA in some risk factors of atherosclerosis, including hypercholesterolemia. Taurine is a semi-essential amino acid that has previously been shown to have endothelial protective effects. The present study was to test whether the protective effect of taurine on endothelial function is related to modulation of the DDAH/ADMA pathway. A single injection of native LDL (4 mg/kg, i.v.) markedly reduced endothelium-dependent vasorelaxation and the plasma level of NO, and increased plasma concentrations of ADMA, malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-alpha). Treatment with taurine in vivo (60 or 180 mg/kg) significantly attenuated the inhibition of endothelium-dependent vasorelaxation and the reduced level of NO, and decreased the elevated levels of ADMA, MDA, and TNF-alpha. Incubation human umbilical vein endothelial cells (HUVECs) with ox-LDL (100 microg/ml) for 24 h markedly increased the medium levels of lactate dehydrogenase (LDH), ADMA, TNF-alpha and MDA, and decreased the level of NO in the medium and the intracellular activity of DDAH. Taurine (1 or 5 microg/ml) significantly attenuated the increases in the levels of LDH, ADMA, TNF-alpha and MDA, and the decrease in the level of NO and the activity of DDAH induced by ox-LDL in HUVECs. The present results suggested that taurine protected against endothelial dysfunction induced by native LDL in vivo or by ox-LDL in endothelial cells, and the protective effect of taurine on the endothelium is related to decrease in ADMA level by increasing of DDAH activity.

Lipoic acid protects gastric mucosa from ethanol-induced injury in rat through a mechanism involving aldehyde dehydrogenase 2 activation.

Numerous studies demonstrate that reactive aldehydes are highly toxic and aldehyde dehydrogenase 2 (ALDH2)-mediated detoxification of reactive aldehydes is thought as an endogenous protective mechanism against reactive aldehydes-induced cell injury. This study aims to explore whether lipoic acid, a potential ALDH2 activator, is able to protect gastric mucosa from ethanol-induced injury through a mechanism involving clearance of reactive aldehydes. The rats received 60% of acidified ethanol through intragastric administration and held for 1 h to establish a mucosal injury model. Lipoic acid (10 or 30 mg/kg) or Alda-1 (a positive control, 10 mg/kg) was given 45 min before the ethanol treatment. The gastric tissues were collected for analysis of gastric ulcer index, cellular apoptosis, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) contents, and ALDH2 activity. The results showed that acute administration of ethanol led to an increase in gastric ulcer index, cellular apoptosis, 4-HNE and MDA contents concomitant with a decrease in ALDH2 activity; these phenomena were reversed by lipoic acid or Alda-1. The gastric protection of lipoic acid was attenuated in the presence of ALDH2 inhibitor. Based on these observations, we conclude that lipoic acid exerts the beneficial effects on ethanol-induced injury through a mechanism involving, at least in part, ALDH2 activation. As a dietary supplement or a medicine already in some countries, lipoic acid can be used to treat the ethanol - induced gastric mucosal injury.

Phosphorylation of Nonmuscle Myosin Light Chain Promotes Endothelial Injury in Hyperlipidemic Rats Through a Mechanism Involving Downregulation of Dimethylarginine Dimethylaminohydrolase 2.

Suppression of dimethylarginine dimethylaminohydrolase (DDAH) activation is related to endothelial dysfunction in hyperlipidemia, and nonmuscle myosin regulatory light chain (nmMLC20) has been show to exert transcriptional function in regulation of gene expression. This study aims to explore whether the suppression of DDAH activation promotes endothelial injury under the condition of hyperlipidemia and whether nmMLC20 can regulate DDAH expression in a phosphorylation-dependent manner. The rats were fed with high-fat diet for 8 weeks to establish a hyperlipidemic model, which showed an increase in plasma lipids and endothelial injury, accompanied by an elevation in myosin light chain kinase (MLCK) activity, phosphorylated nmMLC20 (p-nmMLC20) level, and asymmetric dimethylarginine (ADMA) content as well as a reduction in DDAH2 expression, DDAH activity, and nitric oxide (NO) content. Next, human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low-density lipoprotein (ox-LDL; 100 µg/mL) for 24 hours to establish a cellular injury model in vitro. Consistent with the finding in vivo, ox-LDL induced HUVECs injury (apoptosis and necrosis) concomitant with an increase in MLCK activity, p-nmMLC20 level (in total or nuclear proteins), and ADMA content as well as a reduction in DDAH2 expression, DDAH activity, and NO content; these phenomena were attenuated by MLCK inhibitor. Either in hyperlipidemic rats or in ox-LDL-treated HUVECs, there was not significant change in DDAH1 expression. Based on these observations, we conclude that the suppression of DDAH2 expression might account for, at least partially, the vascular endothelial dysfunction in hyperlipidemia, and nmMLC20 plays a role in suppression of DDAH2 expression in a phosphorylation-dependent manner.

Fenofibrate decreases asymmetric dimethylarginine level in cultured endothelial cells by inhibiting NF-kappaB activity.

Previous investigations have demonstrated that endogenous inhibitors of nitric oxide synthase (NOS), such as asymmetric dimethylarginine (ADMA), contribute importantly to endothelial dysfunction, and that fenofibrate has a protective effect on the endothelium in rats treated with low-density lipoprotein (LDL) by reducing ADMA levels. In the present study, we explored further the possible mechanism underlying inhibition of ADMA generation by fenofibrate in cultured human umbilical vein endothelial cells (HUVECs). Endothelial injury was induced in cultured HUVECs by incubation with oxidative LDL (ox-LDL) and the levels of ADMA, lactate dehydrogenase (LDH), NO and tumour necrosis factor-alpha (TNF-alpha) in the conditioned medium were measured. Cell viability and the activity of dimethylarginine dimethylaminohydrolase (DDAH) and nuclear factor-kappaB (NF-kappaB) in the cultured HUVECs were also determined. Incubation of HUVECs with ox-LDL (100 microg/ml) for 24 h markedly elevated ADMA, LDH and TNF-alpha in the conditioned medium and significantly increased the activity of NF-kappaB, concomitantly with a significant decrease in the activity of DDAH and the content of NO. Pretreatment with fenofibrate (3, 10 or 30 microM) significantly inhibited the increases in ADMA, LDH and TNF-alpha, attenuated the decreased levels of NO and the decreased activity of DDAH and prevented the activation of NF-kappaB. Similar effects were observed in the presence of pyrrolidine dithiocarbamate (PDTC, 10 microM), an antagonist of NF-kappaB. The beneficial effects of fenofibrate on cultured endothelial cells were abolished by MK-886, a specific peroxisome proliferator-activated receptor-alpha (PPARalpha) antagonist. The present results suggest that fenofibrate inhibits ox-LDL-induced endothelial cell damage by decreasing ADMA and increasing DDAH activity, and the protective effects of fenofibrate on endothelial cells may be related to reduction of NF-kappaB activity by activation of the PPARalpha receptor.

Discoidin domain receptors (DDRs): potential implications in atherosclerosis.

Atherosclerosis, one of the most common causes of cardiovascular diseases, is associated with a high morbidity and mortality. It is known that inflammation, vascular smooth muscle cell (VSMCs) phenotypic modulation and atheroma plaque vulnerability are main pathological characteristics of atherosclerosis. The discoidin domain receptors (DDRs), as unique collagen-binding tyrosine kinase receptors, were reported to be involved in the above pathogenesis process of atherogenesis. DDRs were detected on a series of cells within atherosclerotic plaques including macrophages, T cells and smooth muscle cells, and regulated the behaviors of these cells, implicating the potential involvement of DDRs in atherosclerosis. Herein we discuss the roles of DDRs in atherosclerosis, in an attempt to evaluate the value of DDRs as a therapeutic target for atherosclerosis.

A novel function of nuclear nonmuscle myosin regulatory light chain in promotion of xanthine oxidase transcription after myocardial ischemia/reperfusion.

Nuclear myosin regulates gene transcription and this novel function might be modulated through phosphorylation of the myosin regulatory light chain (p-MLC20). Nonmuscle MLC20 (nmMLC20) is also present in the nuclei of cardiomyocytes and a potential nmMLC20 binding sequence has been identified in the promoter of the xanthine oxidase (XO) gene. Thus, we investigated its function in the regulation of XO transcription after myocardial ischemia/reperfusion (IR). In a rat model of myocardial IR and a cardiomyocyte model of hypoxia/reoxygenation (HR) injury, the cardiac or cell injury, myosin light chain kinase (MLCK) content, XO expression and activity, XO-derived products, and level of nuclear p-nmMLC20 were detected. Coimmunoprecipitation (co-IP), chromatin immunoprecipitation, DNA pull-down, and luciferase reporter gene assays were used to decipher the molecular mechanisms through which nmMLC20 promotes XO expression. IR or HR treatment dramatically elevated nuclear p-nmMLC20 level, accompanied by increased XO expression, activity, and products (H2O2 and uric acid), as well as the IR or HR injury; these effects were ameliorated by inhibition of MLCK or knockdown of nmMLC20. Our findings from these experiments demonstrated that nuclear p-nmMLC20 binds to the consensus sequence GTCGCC in the XO gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and hence activates XO gene transcription. These results suggest that nuclear p-nmMLC20 plays an important role in IR/HR injury by transcriptionally upregulating XO gene expression to increase oxidative stress in myocardium. Our findings demonstrate nuclear nmMLC20 as a potential new therapeutic target to combat cardiac IR injury.

Probucol preserves endothelial function by reduction of the endogenous nitric oxide synthase inhibitor level.

1. Oxide low-density lipoprotein (ox-LDL) is believed to play an important role in early events of atherogenesis, and asymmetric dimethylarginine (ADMA) is associated with the development of endothelial dysfunction. The present study examined the effect of a single injection of native low-density lipoprotein (LDL) on endothelium function and the serum level of ADMA and the effect of probucol on endothelium function and ADMA level in rats. 2. Endothelial injury was induced by intravenous injection of LDL at the dose of 2, 4, or 6 mg kg(-1) for 24, 48, or 72 h, and vasodilator responses to acetylcholine in the aortic rings and serum levels of ADMA, nitrite/nitrate (NO) and malondialdehyde (MDA) were determined. 3. Pretreatment with LDL markedly reduced endothelium-dependent relaxation in a concentration-dependent manner. Inhibition of vasodilator responses to acetylcholine by LDL was abolished in the presence of L-arginine (3 x 10(-4) M). Serum levels of ADMA and MDA were significantly elevated in the rats pretreated with LDL, while serum level of nitrite/nitrate was markedly decreased. 4. Pretreatment with probucol significantly improved endothelium-dependent relaxation, decreased concentrations of ADMA and MDA and increased nitrite/nitrate level in the rats treated with LDL. A similar effect was seen in the rats pretreated with an antioxidant vitamin E. 5. These results suggest that a single injection of native LDL causes endothelial dysfunction by elevation of ADMA levels and that the protective effect of probucol on endothelial cells is related to reduction of ADMA concentration.

Reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril: a role of calcitonin gene-related peptide.

Previous studies have shown that the development of tolerance to nitroglycerin is related to a decrease in the release of endogenous calcitonin gene-related peptide (CGRP). In the present study, we explored whether endogenous CGRP is involved in reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril in rats in vivo and vitro. Tolerance was induced by exposure to nitroglycerin (4.4 x 10(-6) M) for 10 min in vitro or by pretreatment with nitroglycerin (10 mg/kg, s.c.) three times a day for 8 days in vivo. Nitroglycerin (3 x 10(-9)-10(-6) M) caused a concentration-dependent relaxation in the isolated rat thoracic aorta, an effect that was reduced by CGRP-(8-37) (3 x 10(-7) M) or capsaicin (3 x 10(-7) M). Preincubation with nitroglycerin for 10 min significantly decreased its vasodilation, which was restored in the presence of N-acetylcysteine (10(-5) M) or captopril (10(-5) M). Nitroglycerin (150 microg/kg, i.v.) produced a depressor effect and an increase in concentrations of nitric oxide and CGRP, and the effects of nitroglycerin disappeared after pretreatment with nitroglycerin for 8 days. However, tolerance to nitroglycerin in vivo also was partially restored in the presence of N-acetylcysteine or captopril. The present results suggest that reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril is related to the increased release of CGRP in the rat.

Protective effects of daviditin A against endothelial damage induced by lysophosphatidylcholine.

Previous investigations have indicated that endogenous inhibitors of nitric oxide synthase (NOS) such as asymmetric dimethylarginine (ADMA) may play an important role in endothelium dysfunction, and some antioxidant drugs improve endothelium function via reduction of ADMA level. The present study examined the antioxidation and endothelial protection of daviditin A, a xanthone compound. Daviditin A significantly inhibited Cu(2+)-induced low-density lipoprotein (LDL) oxidation (EC50: 38.7 microM) and scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals (EC50: 57.5 microM). Vasodilator responses to acetylcholine in rings of the isolated thoracic aorta were impaired in the presence of lysophosphatidylcholine (LPC)(5 mg/l). Daviditin A (10 or 30 microM) significantly attenuated inhibition by LPC of endothelium-dependent relaxation. Incubation of ECV304 cells with LPC (5 mg/l) for 24 h markedly elevated lactate dehydrogenase (LDH) activity and the levels of malondialdehyde (MDA) and ADMA, and decreased the content of nitric oxide (NO) and the activity of dimethylarginine dimethylaminohydrolase (DDAH). Daviditin A (1, 3 or 10 microM) significantly attenuated the increased release of LDH, increased content of MDA, and decreased level of NO induced by LPC. Daviditin A (3 or 10 microM) significantly inhibited the increased concentration of ADMA. Daviditin A (10 microM) significantly attenuated the decreased activity of DDAH. The present results suggest that daviditin A preserves endothelial dysfunction elicited by LPC, and the protective effect of daviditin A on the endothelium is related to reduction of ADMA concentration.

Effect of fenofibrate on LDL-induced endothelial dysfunction in rats.

Previous investigations have demonstrated that asymmetric dimethylarginine (ADMA) is an important factor contributing to endothelial dysfunction, and that fenofibrate has a protective effect on the endothelium in hyperlipidaemic patients. In the present study in rats treated with native low-density lipoprotein (nLDL), we addressed the question of whether the beneficial effect of fenofibrate on endothelial cells is related to reduction of the ADMA concentration. A single injection of nLDL (4 mg/kg, 48 h) markedly reduced endothelium-dependent relaxation in response to acetylcholine and the plasma level of nitrite/nitrate and increased the plasma concentrations of ADMA, malonyldialdehyde (MDA) and tumour necrosis factor-alpha (TNF-alpha). Treatment with fenofibrate (30 or 100 mg/kg) significantly reduced the inhibition of vasodilator responses to acetylcholine, decreased the elevated levels of ADMA, MDA and TNF-alpha, and enhanced the decreased level of nitrite/nitrate in the rats treated with LDL. These results suggest that the protective effect of fenofibrate on endothelial cells in rats treated with LDL may be related to the reduction of ADMA concentration.