Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-β ₂

Abstract

Abstract-In situ analysis of fetal semilunar valve leaflets has revealed cells coexpressing endothelial and mesenchymal markers along the endothelium, with diminished frequency seen in adult valves. To determine whether such cells are progenitor cells, we isolated clonal populations from human pulmonary valves. The clones expressed endothelial markers but showed potential to further differentiate into endothelium in response to vascular endothelial growth factor (VEGF)-A. When exposed to transforming growth factor (TGF)-␤ 2 , individual clones adopted a mesenchymal phenotype to varying degrees and expressed markers of endothelial to mesenchymal transformation (EMT). Both VEGF-and TGF␤ 2 -induced phenotypic changes were partially reversible, indicating the plasticity of these cells. When challenged with VEGF or TGF␤ 2 , a hierarchy of endothelial/mesenchymal potential could be seen among the clonal populations: cells initially closer to an endothelial phenotype showed a strong response to TGF␤ 2 that could be inhibited by VEGF, whereas cells closer to a mesenchymal phenotype responded to TGF␤ 2 but were resistant to endothelialinducing effects of VEGF. These findings suggest the presence of bipotential valve progenitor cells with ability to differentiate into either endothelial or interstitial cells of the valve leaflet. Understanding the differentiation potential and function of these cells may be important for understanding heart valve disease and may also be applied to current paradigms for creating tissue-engineered heart valves. Key Words: cell culture Ⅲ endothelial cell differentiation Ⅲ endothelial cells Ⅲ heart valves Ⅲ progenitor cells Ⅲ transdifferentiation Ⅲ vascular endothelial growth factor Ⅲ vascular endothelium V alvular heart disease is a major cause of morbidity, often resulting in the need to replace the valve with a mechanical or bioprosthetic valve. There is a pressing need for improved approaches, especially for children because calcification and destruction of the replacement valve occurs more rapidly in children and young adults. 1 Possible strategies include stimulating endogenous repair pathways or using autologous progenitor cells to create tissue-engineered heart valves that will grow with the child. For both approaches, an increased understanding of mechanisms underlying the normal cellular turnover and repair throughout adult life will be essential.During embryonic valve development, a subset of endocardial endothelial cells, driven by signals from the underlying myocardium, change their phenotype to mesenchymal cells and migrate into the cardiac jelly to form endocardial cushions, the primordial of valves and cardiac septa of the adult heart. 2 This transdifferentiation of endocardial cells to mesenchymal cells and migration away from the endothelial layer is termed endothelial-mesenchymal transformation (EMT). Studies from Markwald and coworkers show that during EMT, the activated endothelial cells lose cell-cell contacts, gain mesenchymal markers such as ␣-smooth muscle actin (␣-SM...

Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells

“…Caution should, however, be ascribed to characterizing tip cells as strictly endothelial in terms of their structural or functional phenotype. During vasculogenesis as well as angiogenesis, endothelial cells can undergo an endothelial-mesenchymal transformation wherein tissue-invasive cells adopt VSMC-like characteristics, including the expression of ␣-smooth muscle actin (DeRuiter et al, 1997;Frid et al, 2002;Ishisaki et al, 2003;Liebner et al, 2004;Timmerman et al, 2004;Paruchuri et al, 2006). Hence, because endothelial cells engage transcriptional programs necessary to support invasive activity, the leading cell population might be predicted to assume a plastic phenotype more consistent with the unique requirements of the tip cell population.…”

miR-21 represses Pdcd4 during cardiac valvulogenesis

“…Cultivation of HPVECs was as previously described (Paruchuri et al, 2006). miR-21 overexpression was performed by transfection of the miR-21 miRNA precursor (pre-miR miRNA PM12979, Ambion) into HPVECs according to the manufacturer's instructions.…”

“…Lentiviral transduction (Mission shRNA lentiviral TRCN0000059079, Sigma) was performed according to the manufacturer's directions at a MOI of one. TGFβ2 treatment was as described (Paruchuri et al, 2006). Migration assays were performed 6 days after miR-21 transfection and 5 days after TGFβ2 (R&D Systems) treatment, or 6 days after TGFβ2 treatment and 5 days after lentiviral transduction using 6.5 mm Transwells (Costar 8.0 μm pore polycarbonate inserts) as previously described (Paruchuri et al, 2006), with 10% fetal bovine serum as chemoattractant.…”

Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

“…Similar to the heart, pulmonary valve morphogenesis requires EndoMT, which is driven by TGFb2 and is antagonized by the pro-angiogenic factor VEGF-A. 57 Retinal epithelial cells undergo EMT at least in vitro and in vivo during fibrosis that leads to retinopathies, and this EMT is driven by TGFb2, which then induces expression of the Notch ligand Jagged1, that synergistically with TGFb2-Smad and non-Smad signaling promotes the expression of EMTTFs that mediate the mesenchymal transition. 58 In the context of cancer-related EMT, all 3 TGFb isoforms can induce hepatocarcinoma EMT that is mediated by downstream induction and signaling of the platelet derived growth factor (PDGF) A.…”