Aberrant promoter methylation of multiple genes in VSMC proliferation induced by Hcy.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in atherosclerosis (AS), and homocysteine (Hcy) is an independent risk factor for AS. However, the underlying mechanisms are still lagging. Studies have used the combination of methylation of promoters of multiple genes to diagnose tumors, thus the aim of the current study was to investigate the role of methylation status of several genes in VSMCs treated with Hcy. CpG islands were identified in the promoters of platelet­derived growth factor (PDGF), p53, phosphatase and tensin homologue on chromosome 10 (PTEN) and mitofusin 2 (MFN2). Hypomethylation was observed to occur in the promoter region of PDGF, hypermethylation in p53, PTEN and MFN2, and hypomethylation in two global methylation indicators, aluminium (Alu) and long interspersed nucleotide element­1 (Line­1). This was accompanied by an increase in the expression of PDGF, and reductions of p53, PTEN and MFN2, both in mRNA and protein levels. An elevation of S­adenosylmethionine (SAM) and a reduction of S­adenosylhomocysteine (SAH) and the SAM/SAH ratio were also identified. In conclusion, Hcy impacted methylation the of AS­associated genes and global methylation status that mediate the cell proliferation, which may be a character of VSMCs treated with Hcy. The data provided evidence for mechanisms of VSMCs proliferation in AS induced by Hcy and may provide a new perspective for AS induced by Hcy.

High-methionine diets accelerate atherosclerosis by HHcy-mediated FABP4 gene demethylation pathway via DNMT1 in ApoE(-/-) mice.

Homocysteine (Hcy) is an independent risk factor for atherosclerosis, but the underlying molecular mechanisms are not known. We investigated the effects of Hcy on fatty acid-binding protein 4 (FABP4), and tested our hypothesis that Hcy-induced atherosclerosis is mediated by increased FABP4 expression and decreased methylation. The FABP4 expression and DNA methylation was assessed in the aorta of ApoE(-/-) mice fed high-methionine diet for 20weeks. Over-expression of FABP4 enhanced accumulation of total cholesterol and cholesterol ester in foam cells. The up-regulation of DNA methyltransferase 1 (DNMT1) promoted the methylation process and decreased FABP4 expression. These data suggest that FABP4 plays a key role in Hcy-mediated disturbance of lipid metabolism and that DNMT1 may be a novel therapeutic target in Hcy-related atherosclerosis.

Apelin activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas.

Apelin was recently found to be an inotropic polypeptide in isolated rat hearts, and intravenous injection of apelin can induce a transient decrease in blood pressure. To illustrate the mechanism of apelin-induced vasodilation, we observed the in vitro effects of apelin on the L-arginine (L-Arg)/nitric oxide (NO) pathway in the incubated, isolated rat aorta. Apelin stimulated vascular NO(2)(-) product and NOS activation in a concentration- and time-dependent manner. Compared with no apelin treatment, incubation with apelin (10(-9), 10(-8), and 10(-7)mol/L) increased NO(2)(-) product by 33%, 46%, and 69% (all p<0.01), respectively, and Ca(2+)-dependent constitutive NOS (cNOS) activity by 200%, 460%, and 550% (all p<0.01), respectively. However, Ca(2+)-independent NOS (iNOS) activity was not significantly altered (p>0.05). Apelin incubation (10(-9), 10(-8), and 10(-7)mol/L) increased L-Arg uptake by 130%, 180%, and 240% (all p<0.01), respectively. The mRNA level of cationic amino acid transporters, CAT-1 and CAT-2B, in rat aortic tissues treated with 10(-7)mol/L apelin was increased by 110% and 128%, respectively (both p<0.01). Incubation with 10(-7)mol/L apelin elevated eNOS mRNA and protein levels, by 53% (p<0.05) and 319% (p<0.01), respectively. Collectively, these results demonstrate that apelin directly activated the vascular L-Arg/NOS/NO pathway, which could be one of the important mechanisms of apelin-regulated vascular function.

Altered L-arginine/nitric oxide synthase/nitric oxide pathway in the vascular adventitia of rats with sepsis.

1. In recent studies, the vascular adventitia has been established as an important source of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production, even more powerful than the media in response to certain inflammatory factors, such as lipopolysaccharide (LPS). The adventitia has an independent L-arginine (L-Arg)/NOS/NO pathway and is involved in the regulation of vascular function. In the present study, we explored the changes in and the pathophysiological significance of the L-Arg/NOS/NO pathway in the adventitia of rats with sepsis. 2. Sepsis was induced by caecal ligation and puncture in order to observe changes in L-Arg transport, NOS gene expression and activity and NO generation in the vascular adventitia to determine the mechanism of activation of the L-Arg/NOS/NO pathway. 3. Severe sepsis resulted in severe disturbance of haemodynamic features, with decreased mean arterial blood pressure, brachycardia and inhibited cardiac function (decreased left ventricular +/-dP/dt(max)). Left ventricular end-diastolic pressure was elevated threefold (P < 0.01) under anaesthesia. Rats with sepsis showed severe glucopenia and lacticaemia. Plasma levels of the inflammatory factors macrophage chemoattractant protein-1 and interleukin-8 were increased five- and 29-fold, respectively (P < 0.01). 4. In the adventitia of the thoracic and abdominal aortas, the L-Arg/NO pathway was similarly characterized: the uptake of [(3)H]-L-Arg was Na(+) independent, with the peak occurring at approximately 40 min incubation. Total NOS activity was largely calcium independent (> 90%). The V(max) of L-Arg transport in the sepsis group was increased by 83.5% (P < 0.01), but the K(m) value was not significantly different compared with controls. 5. The mRNA levels of cationic amino acid transporter (CAT)-1 and CAT-2B in the sepsis group were increased by 86 and 62%, respectively (both P < 0.01). Inducible NOS activity was increased 2.8-fold compared with controls (P < 0.01) and iNOS mRNA levels were elevated approximately sixfold (P < 0.01). The NO levels in the plasma and incubation media (incubation for 40 min) in the sepsis group were increased by 144 and 273%, respectively (both P < 0.01). 6. The Arg/NOS/NO pathway was activated in the vascular adventitia of rats with sepsis shock. The L-Arg/NOS/NO pathway in the aortic adventitia may play an important role in the pathogenesis of sepsis and septic shock.

Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats.

Intermedin is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family peptide, which has vasodilatory and hypotensive actions identical to those of adrenomedullin and CGRP. Cleavage sites located between 2 basic amino acids at Arg93-Arg94 result in the production of prepro-intermedin95-147, namely intermedin1-53. The bioactive action of intermedin1-53 and its physiological significance are unclear. In this work, we aimed to explore the effects of intermedin1-53 on acute myocardial injury induced by isoproterenol. Myocardial ischemia injury in rats was induced by subcutaneous injection of a high dose of isoproterenol, and the therapeutic effect of intermedin1-53 was observed. Plasma lactate dehydrogenase activity, myocardial and plasma malondialdehyde content were higher in the isoproterenol group than that in controls. Isoproterenol-treated rats showed lower maximal rate of increase and decrease of left-ventricle pressure development (+/-left-ventricle dp/dtmax) and higher left-ventricle end-diastolic pressure (all P<0.01), which suggested severe heart failure and myocardial injury. Semi-quantitative RT-PCR analysis showed that the gene expression of calcitonin receptor-like receptor and receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 in ventricular myocardia were up-regulated by 79% (P<0.01), 48% (P<0.01), 31% (P<0.05) and 130% (P<0.01), respectively, compared with controls. In myocardial sarcolemmal membranes, the maximum binding capacity for [125I]-intermedin1-53 was increased by 118% (P<0.01) in the isoproterenol group compared with controls. Rats treated with low dosage intermedin1-53 (5 nmol/kg/day, 2 days) showed 21% (P<0.05) higher myocardial cAMP content, 18% and 31% higher+left-ventricle dp/dtmax and -left-ventricle dp/dtmax respectively, 288% lower left-ventricle end-diastolic pressure (all P<0.01), and attenuated myocardial lactate dehydrogenase leakage and malondialdehyde formation (all P<0.01). Treatment with high dosage intermedin1-53 (20 nmol/kg/day, 2 days) gave better results than that with low dosage intermedin1-53. These results suggest that the intermedin receptor system was up-regulated in isoproterenol-induced myocardial ischemic injury and intermedin1-53 might play a pivotal cardioprotective role in such injury.

Intermedin1-53 activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas.

Intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family, has similar or more potent vasodilatory and hypotensive actions than adrenomedullin (ADM) and CGRP. The present study was designed to observe the effects of synthetic rat IMD1-53 on L-arginine (L-Arg) cellular transport, nitric oxide synthase (NOS) activity, and nitric oxide (NO) production in the isolated rat aortic ring to illustrate its direct effect on the L-Arg/NOS/NO pathway in vasculature. IMD1-53 significantly increased NO production and cNOS activity in rat aortas and was more potent than equivalent ADM. But the peptides of both IMD and ADM had no effect on inducible NOS expression and activity. Otherwise, IMD1-53 induced a concentration-dependent increase in [3H]L-Arg transport and its effect was more potent than that of an equivalent concentration of ADM. Semiquantitative RT-PCR revealed that IMD1-53 significantly increased cationic amino acid transport (CAT)-1 and CAT-2B mRNA expression, and its effect was similar to that of ADM. All these results suggest that IMD1-53 might regulate vessel function homeostasis via upregulating the L-Arg/NOS/NO pathway.

Apelin protects myocardial injury induced by isoproterenol in rats.

We aimed to explore the change in level of apelin and its receptor APJ during myocardial injury and the therapeutic effects of apelin in myocardial injury. Rat myocardial injury was induced by subcutaneous injection of a high dose of isoproterenol (ISO); apelin and APJ mRNA levels were determined by RT-PCR; APJ protein was determined by Western blot; EIA and RIA were used to measure the apelin content and receptor binding, respectively. Plasma lactate dehydrogenase (LDH) activity and myocardial and plasma malondialdehyde (MDA) contents were higher in ISO-treated hearts than that in controls. ISO-treated rats showed lower +/-LV dp/dt(max) values and higher LVEDP value (all P<0.01), which suggested severe heart failure. As well, the apelin content in plasma, atrial and ventricular myocardium was decreased by 27%, 30% and 25% (P<0.01), respectively. The mRNA levels of apelin and APJ in myocardia were also markedly reduced; but the APJ protein level in myocardia was increased. However, administration of apelin significantly ameliorated myocardial injury and ISO-induced heart failure. Compared with the ISO-alone group, the group given low-dosage apelin (5 nmol/kg/day) had 39% and 66% higher +LV dp/dt(max) and -LV dp/dt(max) values, and 40.7% lower LVEDP value (P<0.01), and the leakage of myocardial LDH and increased MDA content were attenuated (all P<0.01). Interestingly, bolus injections of apelin (10 nmol/kg/day) resulted in potent inotropic effects in ISO-treated rats. ISO-induced myocardial injury resulted in hypoexpression of apelin and its receptor APJ, and the administration of exogenous apelin ameliorated heart failure and myocardial injury. Apelin could have a cardioprotective effect, and the apelin-APJ system may be a new therapeutic target in myocardial injury and heart failure.

Adrenomedullin(27-52) inhibits vascular calcification in rats.

Adrenomedullin (ADM) has the vasodilatory properties and involves in the pathogenesis of vascular calcification. ADM could be degraded into more than six fragments in the body, including ADM(27-52), and we suppose the degrading fragments from ADM do the same bioactivities as derived peptides from pro-adrenomedullin. The present study carries forward by assessing the effects on vascular calcification of the systemic administration of ADM(27-52). The rat vascular calcific model was replicated with vitamin D3 and nicotine. ADM or/and ADM(27-52) were systemically administrated with mini-osmotic pump beginning at seventh day after the model replication for 25 days. Vascular calcific nodules histomorphometry, vascular calcium content, vascular calcium uptake, alkaline phosphatase activity, and osteopontin-mRNA quantification in aorta were assessed. ADM limited 40.2% vascular calcific nodules (P<0.01), did not effect on calcium content (P>0.05), reduced 44.4% calcium uptake (P<0.01), lowered 21.1% alkaline phosphatase activity (P<0.01), and regulated 40.9% downwards osteopontin-mRNA expression (P<0.01) in the aorta of rats with vascular calcification. ADM(27-52) receded 32.0% vascular calcific nodules (P<0.01), taken from 55.5% calcium content (P<0.01), did not affect calcium uptake (P>0.05), inhibited 22.5% alkaline phosphatase activity (P<0.01), and restrained 21.9% osteopontin-mRNA expression (P<0.01) in the aorta of rats with vascular calcification. Both of ADM and ADM(27-52) did interact on vascular calcification each other. ADM could partially antagonize the effects of ADM(27-52) in taking from calcium content (17.5%, P<0.01) and in receding vascular calcific nodules (18.6%, P<0.01). ADM could obviously enhance the action of ADM(27-52) in inhibiting alkaline phosphatase activity (14.4%, P<0.01) and in reducing calcium uptake (11.4%, P<0.01). ADM(27-52) could partially antagonize the effects of ADM on regulating downwards osteopontin-mRNA expression (17.0%, P<0.01). It is concluded that ADM(27-52) derived from ADM acts as an inhibitory agent on vascular calcification, with special mechanisms different from ADM derived from ADM progenitor molecule.

Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts.

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified from human and other vertebrate tissues. Preprointermedin (preproIMD) can generate a 47 amino acid mature peptide (IMD(1-47)) and a shorter 40 amino acid one (IMD(8-47)) by proteolytic cleavage. Amino acid sequence analysis showed that cleavage sites are located between two basic amino acids at Arg93-Arg94, resulting in the production of preproIMD(95-147), namely IMD(1-53). The present study was designed to observe the effects of IMD(1-53) on cardiac function in ischemia/reperfusion (I/R) injury in isolated rat hearts. Perfusion with high-dose IMD(1-53) gave higher left ventricular systolic pressure (LVSP) and maximal rate of increase and decrease of left ventricle pressure (+/-LVdP/dt(max)), and coronary perfusion flow (CPF) than those of controls. Cardiac I/R induced a marked inhibition of cardiac function and myocardial injury. Reperfusion with IMD(1-53) significantly ameliorated the inhibited cardiac function and bradycardia induced by I/R. Compared with the I/R-treatment alone, IMD(1-53) reperfusion augmented CPF, LVSP, and maximal rate of increase and decrease of left ventricle pressure (+/-LVdP/dt(max)) and decreased LVDP. In addition, reperfusion with IMD(1-53)markedly attenuated the leakage of lactate dehydrogenase and malondialdehyde content in myocardia compared with I/R alone. Reperfusion with IMD(1-53)increased the content of cyclic adenosine monophosphate in comparison with I/R alone. Interestingly, the above IMD(1-53) effects are similar to those of adrenomedullin. These results suggest that IMD(1-53), like adrenomedullin, has cardioprotective effects against myocardial I/R injury.

Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts.

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate a 47-amino acid mature peptide (IMD(1-47)) and a shorter 40-amino acid one (IMD(8-47)) by proteolytic cleavage. The present study was designed to determine the protective effect of IMD on cardiac ischemia/reperfusion (I/R) injury and its possible mechanism. Isolated rat hearts were perfused on a Langendorff apparatus and subjected to 45-min global ischemia and 30-min reperfusion. Cardiac function was measured. The release of myocardial protein and lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) were assayed. Myocardial cAMP content was determined by radioimmunoassay (RIA). Cardiac I/R induced a marked inhibition of cardiac function and myocardial injury. Reperfusion with IMD significantly attenuated the I/R injury. Compared with I/R alone, perfusion with 10(-8)mol/L IMD(1-47) and IMD(8-47) induced a 36% and 33% increase in Delta left ventricular pressure (DeltaLVP), 30% and 28% in maximal rate of increase of LV pressure (+LVdP/dt max), and 34% and 31% in maximal rate of decrease of LV pressure (-LVdP/dt max), respectively (all P<0.01) but an approximately 58% and 51% decrease in LV diastolic pressure, respectively (P<0.01). In addition, perfusion with IMD markedly attenuated the leakage of LDH, total protein and myoglobin from myocardia compared with I/R alone. The contents of ventricular myocardia cAMP after reperfusion with 10(-8)mol/L IMD(1-47) and IMD(8-47) were 130% and 91% higher, respectively, than that with I/R alone (all P<0.01). However, formations of myocardial MDA were 52% and 50% lower than that with I/R alone (all P<0.01), respectively. Interestingly, the above IMD effects were similar to those of adrenomedullin (10(-8)mol/L). These results suggest that IMD, like adrenomedullin, exerts cardio-protective effects against myocardial I/R injury.