Abstract
BackgroundTo determine whether and how exosomes from human umbilical vein endothelial cells (HUVEC-Exos) regulates vascular smooth muscle cells (VSMCs) calcification/senescence in high glucose condition.MethodsHUVEC-Exos were isolated from normal glucose (NG) and high glucose (HG) stimulated HUVECs (NG/HG-HUVEC-Exos) by super speed centrifugation. HUVEC-Exos were identified by transmission electron microscopy and Western blot of CD63. Protein profile in HUVEC-Exos was examined to screen the candidate molecules that mediate HUVEC-Exos function. VSMCs were incubated with HUVEC-Exos. A series of functional assays in vitro were performed to assess the effects of HUVEC-Exos on the calcification/senescence of VSMCs. The role of the candidate protein in HUVEC-Exos-induced VSMCs dysfunction was assessed.ResultsExosomes isolated from HG-HUVEC-Exos induced calcification/senescence in VSMCs as assessed by Alizarin Red Staining, senescence-associated β-galactosidase (SA-β-gal) staining, and the expression of ALP and p21. HG-HUVEC-Exos significantly increased LDH activity, as well as the product of lipid peroxidation (MDA content), and decreased oxidative stress marker activity, as compared with NG-HUVEC-Exos. Moreover, mechanism studies showed that mitochondrial membrane potential and the expression levels of mitochondrial function related protein HADHA and Cox-4 were significantly decreased in HG-HUVEC-Exos compared to controls. Proteomic analysis showed that HG-HUVEC-Exos consisted of higher level of versican (VCAN), as compared with NG-HUVEC-Exos. Observation under laser confocal microscopy revealed that most green fluorescence of VCAN could overlap with the red fluorescence came from mitochondria, indicating VCAN is mainly localized to the mitochondria of VSMCs. Knockdown of VCAN with siRNA in HUVECs, inhibited HG-HUVEC-Exos-induced mitochondrial dysfunction and calcification/senescence of VSMCs.ConclusionsOur data indicate an intracellular role for VCAN in VSMCs. VCAN participates in hyperglycemia-induced calcification/senescence via modulation of mitochondrial function in VSMCs.
Highlights
To determine whether and how exosomes from human umbilical vein endothelial cells (HUVEC-Exos) regulates vascular smooth muscle cells (VSMCs) calcification/senescence in high glucose condition
high glucose (HG)‐HUVEC‐Exos induce VSMCs calcification/senescence To determine the ability of HUVEC-Exos in regulating calcification/senescence in hyperglycaemic condition, VSMCs were cultured in the presence of HUVEC-Exos
We found that compared with normal glucose (NG)-HUVEC-Exos, HGHUVEC-Exos induced calcification/senescence of VSMCs as determined by Alizarin Red Staining and SA-β-gal staining, respectively (Fig. 2a, b)
Summary
To determine whether and how exosomes from human umbilical vein endothelial cells (HUVEC-Exos) regulates vascular smooth muscle cells (VSMCs) calcification/senescence in high glucose condition. Diabetes and associated complications give rise to a tremendous burden on the healthcare and present major challenges to patients and national economies. Morbidity and mortality of diabetic patients are substantially aggravated by vascular complications including coronary artery, cerebrovascular, and peripheral artery disease. Vascular calcification/aging could influence the threshold, process, severity and prognosis of diabetic vascular complications [2, 3]. A major determinant of vascular aging is vascular calcification, characterized by vascular smooth muscle cells (VSMCs) calcification (Monckeberg’s calcification). There is accumulating evidence suggesting that VSMCs calcification/senescence have central roles in the development and progression of diabetes-related cardiovascular disorders [4, 8]
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