Inflammatory Cytokines Promote Mesenchymal Transformation in Embryonic and Adult Valve Endothelial Cells

Abstract

Objective Inflammatory activation of valve endothelium is an early phase of aortic valve disease pathogenesis, but subsequent mechanisms are poorly understood. Adult valve endothelial cells retain the developmental ability to undergo endothelial to mesenchymal transformation (EndMT), but a biological role has not been established. Here we test whether and how inflammatory cytokines (TNF-α and IL-6) regulate EndMT in embryonic and adult valve endothelium. Methods and Results Using in vitro 3D collagen gel culture assays with primary cells, we determined that IL-6 and TNF-α induce EndMT and cell invasion in dose dependent manners. Inflammatory-EndMT occurred through an Akt/NFκB-dependent pathway in both adult and embryonic stages. In embryonic valves, inflammatory-EndMT required canonical TGFβ signaling through Alk2/5 to drive EndMT. In adult valve endothelium, however, inflammatory-induced EndMT still occurred when Alk2/5 signaling was blocked. Inflammatory receptor gene expression was significantly upregulated in vivo during embryonic valve maturation. Endothelial-derived mesenchymal cells expressing activated NFκB were found distal to calcific lesions in diseased human aortic valves. Conclusions Inflammatory cytokine induced EndMT in valve endothelium is present in both embryonic and adult stages, acting through Akt/NFκB but differently utilizing TGFβ signaling. Molecular signatures of valve EndMT may be important diagnostic and therapeutic targets in early valve disease.

Oxidative stress mediates the conversion of endothelial cells into myofibroblasts via a TGF-β1 and TGF-β2-dependent pathway

“…34,35 In addition, tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), and IL-1b also induce the EndMT. 41,56,57 Furthermore, we recently demonstrated that the endotoxin lipopolysaccharide induces the acquisition of fibrotic-like phenotype in ECs. 37 This conversion rises as a potential mechanism that begins endothelial dysfunction during the inflammatory process.…”

“…(vi) Furthermore, we used RT-qPCR and protein detection by western blot to demonstrate that H 2 O 2 induces the expression and secretion of TGF-b1 and TGF-b2, and it has been clearly demonstrated that TGF-b1 and TGF-b2 induce the conversion of ECs into myofibroblasts. 19,34,35,40,41,85 expression by converting them from normal to pathological. This information will be useful in designing new and improved therapeutic strategies against oxidative stressmediated systemic inflammatory diseases.…”

“…However, by means of EndMT, a fibrotic-like spindle-shaped phenotype with non-connected cells is observed along with a-SMA and FSP-1 overexpression, which alters the cytoskeletal organization. [34][35][36][37][38][39][40][41][42][43] Furthermore, acquisition of ECM proteins such as fibronectin and collagen type III is a prominent functional feature of myofibroblasts that changes endothelial functionality. Healthy ECs secrete collagen type IV and low amounts of fibronectin, whereas collagen type I and type III are virtually absent, appearing only after fibrosis has been established and affecting normal endothelial function.…”

“…The increase in ECM proteins during EndMT alters EC function because it affects the interaction between ligands and membrane receptors, protein turnover, and protein internalization. 34,36,37,[41][42][43][44][45][46][47] In addition, increased cell migration is a major distinctive feature of myofibroblasts. Because EndMT is a cellular mechanism for the conversion of polarized ECs into motile mesenchymal cells, this process is also characterized by the acquisition of migratory features.…”

Comparison of Mesenchymal Stem Cell Source Differentiation Toward Human Pediatric Aortic Valve Interstitial Cells within 3D Engineered Matrices

“…28 Tissue was procured with consent as approved by the Institutional Review Board of WeillCornell Medical College in New York City as previously described. 29 Cells were cultured in HAVIC growth medium (HGM) containing MCDB131 medium (Sigma), 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin/streptomycin (P/ S; Invitrogen), 0.25 mg/L recombinant human fibroblast growth factor basic (rhbFGF; PeproTech), and 5 mg/L recombinant human epidermal growth factor (rhEGF; Invitrogen). 28,30,31 Cells were used at passages 4-8.…”