Songming Chen

Expression of heme oxygenase-1, hypoxia inducible factor-1α, and ubiquitin in peripheral inflammatory cells from patients with coronary heart disease

Abstract Background: The increased expression of heme oxygenase-1 content, a stress-response protein, directly correlates with the incidence of coronary heart disease. Down-regulation of hypoxia inducible factor-1α activity, a major downstream effector of the signaling pathways activated by hypoxia, increases cell survival after hypoxia. The ubiquitin system, a non-lysosomal pathway of protein degradation, is involved in processes of coronary heart disease. The aim of this study was to investigate the expression of heme oxygenase-1, hypoxia inducible factor-1α, and ubiquitin in both monocytes and lymphocytes isolated from patients at the mRNA and protein levels in different stages of coronary heart disease and their possible correlation. Methods: A total of 90 patients with coronary heart disease (30 acute myocardial infarction, 30 unstable angina pectoris, and 30 stable angina pectoris) were selected, and 30 cases with normal coronary artery served as controls. The mRNA and protein expression of heme oxygenase-1, hypoxia inducible factor-1α, and ubiquitin in monocytes and lymphocytes were examined by semi-quantitative reverse transcriptase polymerase chain reaction and Western blotting, respectively. Results: The mRNA expression of heme oxygenase-1 and ubiquitin was associated with the severity of coronary heart disease (p<0.05). There was no significant difference in hypoxia inducible factor-1α mRNA expression between the coronary heart disease patients and controls. The protein expression of heme oxygenase-1, hypoxia inducible factor-1α, and ubiquitin was significantly stronger in patients with coronary heart disease than in controls, and the expression levels increased with the severity of the disease. There was a positive association between heme oxygenase-1 and hypoxia inducible factor-1α and ubiquitin, antioxidative therapy with adrenergic receptor blocker, angiotensin-converting enzyme inhibitor or statins up-regulated the expression of heme oxygenase-1 and hypoxia inducible factor-1α. Conclusions: These data suggest that heme oxygenase-1, hypoxia inducible factor-1α, and ubiquitin are involved in the development and progression of coronary heart disease and thus may be useful biomarkers for coronary heart disease. Clin Chem Lab Med 2009;47:327–33.

Haem oxygenase-1 expression and coronary heart disease--association between levels of haem oxygenase-1 expression and angiographic morphology as well as the quantity of coronary lesions.

Objective — The aim of this paper was to investigate the association between levels of HO-1 expression and angiographic morphology as well as the quantity of coronary lesions in patients with coronary heart disease (CHD). Methods — 110 patients with CHD were diagnosed by coronary angiography which contained coronary lesions in some way. Firstly, the patients were divided into 3 groups according to the angiographic morphology of their coronary lesions: type I (smooth borders) group (n1= 36), type II (irregular borders) group (n2= 48) and type III (long and irregular lesions) group (n3= 26). Secondly, the patients were split into a further 3 groups, named: single-vessel group (SV, 38 cases), double-vessel group (DV, 44 cases) and multi-vessel group (MV, 28 cases) according to the number of coronary lesions. Another 30 patients with normal coronary arteries (diagnosed by coronary angiography) were selected as the control group. The levels of HO-1 protein expression in monocytes and lymphocytes from the subjects were tested by immunohistochemistry and Western blot. A computer picture analysing system was also used to measure the levels of HO-1 protein expression. Results — HO-1 protein was located in cell plasma and the levels of HO-1 protein expression in patients with CHD were significantly higher than in those without CHD (p < 0.01).There were significant differences of HO-1 expression among patients with CHD. Patients with type III lesions had the highest levels, followed by those with type II lesions and the lowest levels were found in patients with type I lesions (p < 0.01). Also, levels of HO-1 protein expression in patients with multi-vessel disease and double-vessel disease were significantly higher than in those with single-vessel disease (p < 0.01). Conclusions — There is a higher expression of HO-1 in patients with CHD and the levels of HO-1 protein are associated with severity of CHD angiographically.

Serum ubiquitin via CXC chemokine receptor 4 triggered cyclooxygenase-1 ubiquitination possibly involved in the pathogenesis of aspirin resistance

Extracellular ubiquitin (Ub) with platelet aggregation property was found higher in acute myocardial infarction (AMI) patients. Here we detected the platelet functions and serum Ub levels in 250 AMI patients and 50 healthy volunteers before and after aspirin treatment. The influence of serum Ub on platelet functions was determined in vitro. We found that 47 out of 250 AMI patients showed aspirin resistance (AR) and 203 showed aspirin sensitivity (AS). During hospitalization, AR group had higher serum Ub levels than the AS group or the healthy group, and the serum Ub levels was related to the rates of thrombosis events. The patients with higher serum Ub levels showed that the platelets had more ubiquitinated platelets, higher contents of ubiquitinated proteins and ubiquitinated cyclooxygenase-1 (COX-1). The levels of ubiquitinated COX-1 in the platelets was inversely correlated with acetylated COX-1, the separated ubiquitinated COX-1 activity was approximately twofold or fourfold higher than the total COX-1(ubiquitinated COX-1 and COX-1) or COX-1. In vitro, we found that extracellular Ub, via the CXC chemokine receptor 4 (CXCR4) pathway, facilitated COX-1 to be ubiquitined and prevented aspirin to acetylate its target. Platelets had higher levels of ubiquitinated COX-1 showing poor response to aspirin. Such results were not detected in Ub-free serum or ovalbumin incubated platelets. Serum Ub, via the CXCR4 pathway, facilitated COX-1 to be ubiquitined and activated the platelets possibly involved in the pathogenesis of AR.

Chronic aspirin via dose-dependent and selective inhibition of cardiac proteasome possibly contributed a potential risk to the ischemic heart

Impaired cardiac proteasome has been reported in ischemic heart and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, its unclear whether it affects cardiac proteasome functions. Myocardial infarction (MI), sham or normal male SD rats were injected intraperitoneally with high (300 mg/kg), low (5 mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated human ventricular myocytes. Dose-related increases in heart and ventricular weight, and impaired cardiac functions, were found more exacerbated in the aspirin-treated MI rat hearts than the saline-treated MI counterparts. The activity of 26S, 20S and 19S declined by about 30%, or the 20S proteasome subunits β5, β2 and β1 decreased by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats (P<0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, β5 by 30% and 12%, and β1 by 40% and 30%, respectively, and the lost activity was correlated with the compromised cardiac functions or the decreased cell viability. The dose-related and selective inhibition of 26S and 20S proteasome, or the 20S proteasome subunits β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. The impaired cardiac proteasome, enhanced by chronic aspirin treatment, attenuated the removal of oxidative and ubiquitinated proteins, and chronic aspirin treatment via selective and dose-dependent inhibition of cardiac proteasome possibly constituted a potential risk to ischemic heart.

Germanium in ginseng is low and causes no sodium and water retention or renal toxicity in the diuretic-resistant rats.

Ginseng preparations contain high concentrations of germanium (Ge), which was reported to contribute to diuretic resistance or renal failure. However, Ge content in ginseng and the influence on renal functions remain unclear. Forty rats were randomly divided into control group, low, moderate, and high Ge ginseng-treated group and observed for 25 days. Daily urine, renal functions, and serum and urine electrolytics were measured. Ge retention in the organs and renal histological changes were also evaluated. Ge content ranged from 0.007 to 0.450 µg/g in various ginseng samples. Four groups showed no difference in the daily urine output, glomerular filtration rate, urinary electrolytes excretions, 24 h-urine protein, as well as plasma and urine urea nitrogen, creatinine, osmotic pressure, and pH values. Ge did not cause any renal pathological effects in this study. No Na and water retention was detected in the ginseng-treated groups. Ge retention in various organs was found highest in spleen, followed by the kidney, liver, lung, stomach, heart, and pancreas. The total Ge contents in various ginsengs were low, and ginseng treatment did not affect renal functions or cause renal histological changes.

Elevated survivin expression in peripheral blood mononuclear cells is central to collateral formation in coronary chronic total occlusion

Survivin is essential to angiogenesis and revascularization, but its role in coronary collateral formation remains unclear. The role of survivin in peripheral blood mononuclear cells (PBMCs) of coronary chronic total occlusion (CTO) patients was investigated. Coronary CTO patients (n=46; mean age 60.1±8.5, male 54.3%) (CTO group) and normal control patients (n=18; mean age 58.0±10.0, male 55.6%) underwent angiographic collateral vessel grading by Rentrop classification (C0 – C3) and provided peripheral blood between June 2006 and February 2007. Rat hind limb ischemia models were constructed using four equal groups of Sprague-Dawley rats (n=36): normal control, sham operation, operation and granulocyte macrophage colony-stimulating factor (GM-CSF). PBMC numbers and characteristics, collateral vessels, survivin, CD4, CD8, CD44, vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) expression were determined using RT-PCR, flow cytometry, immunocytochemistry and western blot analysis. PBMC survivin mRNA and protein expression levels were higher in patients with good collateral circulation (C2 + C3) than in patients with no collateral flow (C0) (all P<0.05). Survivin single-positive and survivin and CD8, VEGF and ICAM-1 double-positive percentages were elevated in patients with good collateral circulation compared to those with normal and no collateral flow (all P<0.05), consistent with the rat model results, wherein higher survivin levels produced significantly larger and more visible collateral vessels. In conclusion, elevated survivin expression in PBMCs, particularly survivin and CD8, VEGF, and ICAM-1 double-positive PBMCs, may be crucial for good collateral formation in patients with coronary CTO, as confirmed by assessment of a rat model.

EXTRACELLULAR UBIQUITIN VIA CXC CHEMOKINE RECEPTOR 4 ENHANCES PLATELET ACTIVITY BY UBIQUITINATION OF CYCLOXYGENASE-1

Objectives To investigate the mechanism of extracellular ubiquitin (Ub) influence on the platelet functions. Methods The arachidonic acid (AA)-preincubated healthy platelets were treated with different concentrations of extracellular Ub (50, 100, 500 and 1000 ng/ml), or AMD3100 (50, 100, 500 and 1000 ng/ml) (antagonist of CXCR4) prior to extracellular Ub (1000 ng/ml)). Platelet functions were determined by light-transmission platelet aggregometry (LTA) or thrombelastography (TEG) platelet mapping. The protein expressions of CXCR4 and ubiquitinated proteins, including ubiquitinated COX-1, were detected by western blot or Immunoprecipitation (IP). Results LTA or TEG showed that the activity of platelet exposed to extracellular Ub at 100, 500 and 1000 ng/ml was significantly increased compared with that at 50ng/ml. The increased activity was positively correlated with the levels of ubiqitinated proteins in the platelets. However, AMD3100 dose dependently counteracted the effect of Ub, and the platelet aggregation decreased accordingly. Further, CXCR4 could be activated by extracellular Ub or inhibited by AMD3100 dose dependently, and the level of CXCR4 was correlated with the ubiquintinated proteins, including ubiquitinated COX-1, in the platelets. IP experiments showed that the ubiqitinated proteins in the platelets contained ubiquitinated COX-1, whose levels were correlated with the expressions of ubiqitinated proteins. Inhibition of CXCR4 by AMD3100 caused a dose-related decline in ubiquitinated COX-1, which was correlated positively with platelet activity. Conclusions The data suggested that extracellular Ub dose dependently, via CXCR4 pathway, facilitated its internalisation into the platelet to enhance the ubiquitination of COX-1, which contributed to the increase in platelet activity.

Expression of Ubiquitin in Peripheral Inflammatory Cells from Patients with Coronary Artery Disease

In addition to its role in the removal of damaged and unneeded proteins, the ubiquitin–proteasome system (UPS) may play a key role in coronary artery disease (CAD). To investigate the expression of ubiquitin in monocytes and lymphocytes isolated from patients at different stages of CAD, 120 patients with CAD (40 with acute myocardial infarction [AMI], 40 with unstable angina pectoris [UAP] and 40 with stable angina pectoris [SAP]) were selected; 40 patients with normal coronary arteries served as controls. At both the mRNA and protein levels, ubiquitin expression was higher in patients with CAD than in controls, and patients with AMI had a much higher expression of ubiquitin (at both the mRNA and protein levels) than those with SAP and UAP. These data indicate that ubiquitin expression increased with increasing severity of CAD, suggesting that ubiquitin may play a critical role in the development and progression of CAD.