Study On AOPPs Induce Monocyte Chemoattractant Protein-1 Expression And Its Passway In Vascular Smooth Muscle Cells

BackgroundCardiovascular diseases are the most common complications seen in patients with end-stage renal disease (ESRD), whose death rate were 10-20 fold as high as the general population.Enough developing dialysis treatment technic markedly decreased the incidence of acute events threatened to ESRD, however, the death rate due to cardiovascular diseases(CVD) was still high in ESRD. Most of the traditional CVD risk factors, such as older age, diabetes mellitus, systolic hypertension, left ventricular hypertrophy(LVH), and low high-density lipoprotein (HDL) cholesterol, are highly prevalent in ESRD, but which can not explain why such a high morbidity and mortality of CVD. It is very important to further find what causes to lead to promote the CVD in uremia.Advanced oxidation protein products (AOPP) are dityrosine-containing protein cross-linking products isolated from the plasma of hemodialysis (HD) patients, which was firstly reported by Witko-Sarsat in 1996. AOPP represent a generic name for final products of oxidative modification of proteins caused by oxidative stress in HD patients. AOPP rise as novel uremic toxins with the progression of chronic renal failure (CRF) and oxidative stress and accumulate in patients'plasma and tissues. It has been clarified that AOPP are a novel class of pro-inflammatory mediators with monocytes being their elective cellular target. AOPP are able to trigger the oxidative burst of monocytes and their release of inflammatory cytokines, and join the process of systemic micro-inflammation in CRF patients, which is a crucial pathogenic link to long-term uremic complications, such as dysregulation of immune system, accelerated atherosclerosis and dialysis-related amyloid arthropathy.It has been reported 3that AOPPs is associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. little is known about the direct effects of AOPPs on vascular smooth muscle cells (VSMCs).Monocyte-derived macrophages play an important role throughout all stages of atherosclerosis. From the initial events to the advanced lesions, the presence of the monocyte-macrophages reinforces the notion that plaque pathogenesisinvolves strong inflammatory components. Monocyte chemoattractant protein-1 (MCP-1), a member of C–C chemokines, stimulates the migration of monocytes through an arterial endothelial four monolayer and is postulated to play a critical role for the development of atherosclerosis . MCP-1 can be expressed in a variety of cells including monocytes, smooth muscle cells, and endothelial cells and is also observed in atherosclerotic lesions .In this study, we investigated whether MCP-1 production is stimulated in VSMCs by AOPPs. Moreover, we explored the signal transduction pathway involved in AOPPs-induced MCP-1 expression. To search for new interference targets to block their toxic effects,we also investigated the effect of preconditioning high dosage of vitamin C for the dialyzer and the effect of the mode of hemopurification on AOPP in hemodialysis patients.This will help to seek for possible interference targets to block toxic effects of AOPP and build a solid basis for future study of new clinical treatment that is able to prevent and cure CRF complications.Results1. AOPP-HSA and AOPP-BSA stimulated MCP-1 production and secretion with a dose- and time-dependent manner. Results of western blot showed that MCP-1 protein expression continued to increase for up to 24 hours with 200μmol/L of AOPP. This stimulatory effect of AOPP was observed at the concentrations of 400μmol/L of AOPP, but there is no significance compared with that of 200μmol/L.2. AOPP-HSA and AOPP-BSA stimulated MCP-1 mRNA expression with a dose- and time-dependent manner. MCP-1 mRNA expression was detected on VSMCs with no stimulant. The stimulating effects increased while the doses of AOPP increased. An increase in MCP-1 mRNA expression was detected after 1h incubation with 200μmol/L AOPP, was maximum at 4 hours, and stopped at 24 hours. MCP-1 mRNA was induced maximally at the concentrations of 200μmol/L .3. To verify the specificity of AOPP effect, we observed the effect of 2 different AOPP modified proteins (AOPP-HSA and AOPP-BSA) and 2 different unmodified proteins (HSA and BSA) on MCP-1 expression in the experiments of western blot and RT PCR and found that the unmodified proteins had no effect on MCP-1 expression. Meanwhile, there was no difference between the stimulating effect of AOPP-HAS and that of AOPP-BSA. These evidences implied that the inhibitory effect of AOPP modified proteins on MCP-1 expression was due to AOPPs but not the proteins. To exclude the interference of endotoxin, we observed the affect of PMX-B, a chelator of endotoxin, on the stimulating effect of AOPP modified proteins. Results showed that the PMX- B didn't abate or abolish the promoting effect of AOPP modified proteins on MCP-1 expression. This meant that the above results are not interfered by endotoxin.4. Simultaneously with the observation of AOPP modified proteins promoting MCP-1 production by VSMCs, we also found the enhanced p38-MAPK activity with a dose- and time-dependent manner relating to AOPP modified proteins. The stimulation of AOPP modified proteins on MCP-1 production was blocked by SB203580, the specific inhibitor of p38-MAPK. These evidences implied that AOPP modified proteins stimulating MCP-1 production by endothelial cells through the p38-MAPK signal pathway.5. Serum levels of AOPP were significantly higher in uremia patients compared with healthy controls,while higher in HD patients than in uremia patients(without HD), but significantly lower in therapy groups(preconditioning high dosage of vitamin C for the Dialyzer) than in HD groups. Serum levels of AOPP were significantly lower in HDF and HP patients compared with before treatment,while having no significantly changes in HD patients .But after half-year treatment the serum levels of AOPP were significantly higher than before in HD groups,still lower in HDF patients and having no significantly changes in HP patients.Conclusions1. AOPP may be involved in the progression of atherosclerosis by promoting MCP-1 protein expression and up-regulate MCP-1mRNA in VSMCs.2. the AOPP modification-induced pathobiological cascades were mediated by p38-MAPK, and inhibitor of p38-MAPK may be a new way to prevent the cardiovascular diseases caused by CRF. 3. Clinical observation discovered that long-term HDF treatment and preconditioning high dosage of vitamin C for the Dialyzer may also be a kind of methods to prevent the cardiovascular diseases in HD patients.