ABSTRACT Patients with chronic kidney disease (CKD) have high cardiovascular mortality, and this could be partly due to the development of vascular calcification, which is characterized by a pathological deposition of calcium and phosphate in the arterial walls. Vascular calcification is common in patients with CKD due to the accumulation of uremic toxins and metabolic disturbances such as hyperphosphatemia. Unfortunately, there is no effective treatment to prevent or slow the progression of vascular calcification. Our overarching aim is to identify new therapeutic targets to treat vascular calcification by studying the interaction between two major vascular cell types—endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). In the pathogenesis of vascular calcification, while osteogenic differentiation of VSMCs is the key process, EC-VSMC interaction also appears to be important, yet EC-VSMC interaction is not well-studied. To address this knowledge gap, we have assembled a multidisciplinary team of experts and established an insert co-culture system of primary human aortic ECs and human aortic VSMCs. Compared to mono-culture, this co-culture system allows us to model distinct aspects of the multi-cellular environment in vivo. We will test the central hypothesis that uremic serum from patients with severe CKD alters EC secretome, which in turn induces osteogenic differentiation of VSMCs and calcification. Studying uremic serum takes into account the complex metabolic alteration in CKD and has great translational potential. Our two aims are (1) to define the paracrine effects of ECs on phosphate induced calcification of VSMC culture; (2) to determine the effects of uremic serum via EC-VSMC signaling on osteogenic differentiation of VSMCs and calcification. In Aim 1, using medium containing high phosphate, VSMCs will be cultured with or without ECs, or with ECs transfected with small interfering RNA targeting phosphate transporters to block the intracellular uptake of phosphate. Then, we will elucidate the role of the nitric oxide pathway in EC-VSMC paracrine signaling and identify novel signaling pathways by examining cellular secretome using an unbiased approach of SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics. In Aim 2, VSMCs will be cultured with or without ECs, and in the presence of uremic or normal serum. Uremic serum will be obtained from patients with end stage kidney disease receiving hemodialysis and compared to normal serum from age- and sex-matched healthy volunteers. Then, we will quantify cellular secretome using SILAC-based proteomics to identify specific paracrine factors, and fractionate serum using chromatography to isolate the components of uremic serum that promote calcification. Studying EC-VSMC signaling will help identify new therapies that can attenuate vascular calcification, making this proposal significant. With the use of human uremic serum, primary human cells in an insert co-culture system, and SILAC-based proteomics, this project is highly innovative and has great translational potential.
|Effective start/end date||9/1/22 → 6/30/23|
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