Monocytes,VEGF-C,IL-6,hs-CRP,FIB And D-D Are Associated With Severity Of Human Peripheral Artery Stenosis

Background:Peripheral arterial disease (PAD) is a manifestation of systemic atherosclerosis. It affects 10-15% of the general population, and approximately 50% of PAD patients are asymptomatic, leading to under-diagnosis and under-treatment of the disease.Previous studies support that inflammation and innate immune cells including monocytes or macrophages play important roles during the development and progression of peripheral artery stenosis (PAS). Recently, we reported that serum vascular endothelial growth factors-C (VEGF-C) expression was substantially increased in sepsis patients, suggesting serum VEGF-C as a new biomarker for sepsis. A link has been established between pathogenesis of atherosclerosis and the VEGF family members. Although we provided evidence that serum VEGF-C levels are associated with human or murine sepsis, it remains unknown whether VEGF-C is linked to PAS severity. Various risk factors are used as potential criteria for detection of subclinical PAS, such as inflammation biomarkers including C-reactive protein, interleukin-6 (IL-6) fibrinogen (FIB), lipoprotein, cholesterol, along with blood glucose and triglyceride, it remains unknown whether these risk factors are linked to PAS severity.PAD is associated with significant morbidity and mortality. Although convention risk factors contribute to the onset and progression of PAD, the role of biomarkers in pathways of inflammation and thrombosis in determining susceptibility to PAD and PAS is being increasingly recognized. Novel risk factors for PAD and severe PAS patients may allow earlier detection, an improved understanding of disease etiology and progression, and the development of new therapies.Objective:Emerging reports propose possible biomarkers that are related to inflammation, coagulation, nutrition and lipid parameters for detection of the progression of atherosclerotic plaques, peripheral artery disease, particularly peripheral artery stenosis. However, it remains unclear which biomarkers in serum are associated with the severity of PAS.Methods:In this study, we measured serum levels of inflammatory biomarkers along with lipid and nutritional parameters in 27 control subjects and 74 patients who suffered different degrees of PAS. Thrombelastography (TEG) and regular coagulation function were tested in 27 control subjects and 53 patients who suffered different degrees of PAS. Peripheral blood mononuclear cells(PBMCs) from control subjects and PAS patients were isolated and immunostained with anti-CD11b and anti-F4/80 antibodies. Total RNA was isolated from monocytes then converted to cDNA, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed. The average mRNA levels of IL-6 and VEGF-C in monocytes from control subjects and PAS patients were calculated. Monocytes were seeded into 96-well tissue culture plates at a density of 1.0×105 per well and stimulated with or without lipopolysaccharide (LPS) (100 or 300 ng/mL) for 24h in DMEM. Concentrations of IL-6 and VEGF-C from supernatants of fresh isolated monocytes or LPS-treated monocytes were detected by ELISA. Number of monocytes from PAS patients that were adherent to fibrinogen was compared with those from control subjects. Phagocytosis ability to clear lipid of monocytes was compared between two groups too.Results:Serum concentrations of IL-6, high sensitive C-reactive protein (hs-CRP), low density lipoproein(LDL), and cholesterol were significantly increased in patients showing moderate or severe PAS. Serum concentration of VEGF-C was significantly increased in patients showing severe PAS. Compared with control subjects, the levels of serum fibrinogen and D-dimer (D-D) were higher in patients showing moderate and severe PAS. TEG test showed that compared with control subjects, R value and K value were significantly shorter in patients showing moderate and severe PAS, but Angel a value and MA value were larger. Furthermore, numbers of blood monocytes, leukocytes and neutrophil from moderate or severe PAS patients were significantly increased. Monocytes from PAS patients showed elevated adhesion to plate-coated fibrinogen and phagocytosis ability to clear lipid. Compared to control subjects, freshly isolated or LPS-stimulated blood monocytes from PAS patients displayed enhanced ability to produce VEGF-C and IL-6 at high levels.Conclusion:Our study suggests that the increased numbers of blood monocytes might play key roles during the development of severe PAS, which enhance adhesion at the local narrowed peripheral artery and secret high levels of VEGF-C and IL-6. We suggest that serum concentrations of VEGF-C might be used as biomarkers for diagnosis severe PAS in combiantion with clinical imaging examination. In addition, IL-6, hs-CRP, FIB and D-D maybe contribute to the onset and progression of PAS. TEG indexes reflected that coagulation function was abnormal in patients with moderate and severe PAS.