Uraemic extracellular vesicles augment osteogenic transdifferentiation of vascular smooth muscle cells via enhanced AKT signalling and PiT‐1 expression

Abstract

Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu‐stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. ‘Uraemic’ and control EV (EVUR; EVCTRL) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro‐calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EVCTRL and EVUR differed in size and miRNA contents. Contrary to EVCTRL, EC‐ and plasma‐derived EVUR significantly increased the pro‐calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22α. Further, EVUR enhanced the protein expression of the phosphate transporter PiT‐1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT‐1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro‐calcifying effects of EVUR. Similar effects were achieved by inhibition of miR‐221/‐222 and mimicking of miR‐143/‐145 in EVUR. In conclusion, EVUR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD.

RNA-seq analysis of extracellular vesicles from hyperphosphatemia-stimulated endothelial cells provides insight into the mechanism underlying vascular calcification

“…Our current results showed that miR-143-3p was remarkably down-regulated in HP-EC-EVs. In consistent with our results, miR-143 expression in extracellular vesicles (EV) derived from urea and indoxyl sulphate-stimulated EC (EV UR ) were also down-regulated, and mimicking of miR-143 in EV UR blocked the pro-calcifying effects of EV UR [ 21 ]. Among those miRs, the miR-30 family has been reported to play an important role in osteogenesis [ 22 ].…”

Extracellular Vesicles as Drivers of Immunoinflammation in Atherothrombosis

“…EVs from dysfunctional endothelium [ 164 ] and senescent ECs [ 158 , 165 ] promote vascular calcification. VSMCs also release calcifying EVs themselves [ 166 , 167 ]. One described mechanism driving the VSMCs towards calcification involves the regulation of calcifying EVs by sortilin [ 166 ].…”

Uremic Toxin Lanthionine Induces Endothelial Cell Mineralization In Vitro

“…In the context of CKD, dangerous signals from uremic toxins and cytokines may induce various molecular mechanisms, leading to injury. Studies demonstrated that uremic toxins stimulated ECs to produce extracellular vesicles (EV), promoting the calcium and phosphate effects of adjacent cells [ 53 ]. Dysfunction and injury of ECs were associated with various diseases, such as cardiovascular diseases (CVDs), cancer and kidney disease.…”