Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression

Abstract

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats.

Nestin expression is upregulated in the fibrotic rat heart and is localized in collagen-expressing mesenchymal cells and interstitial CD31(+)- cells

“…However, hypertrophy alone was not sufficient to induce nestin expression in neonatal and adult cardiomyocytes. [11,23] However, concentric remodeling of the heart was associated with ischemic injury and may represent the incipient event promoting in part the subsequent reactive fibrotic response. [25] Thus, the appearance of nestin (+) -cardiomyocytes in the pressure-overloaded hypertrophied rat heart may be secondary to ectopic ischemic injury.…”

“…[1,2] Nonetheless, the latter paradigm was challenged by several recent studies demonstrating that the majority of collagen type I (+) -mesenchymal cells identified in the interstitial and perivascular regions of the fibrotic pressure-overloaded mouse heart and in the fibrotic rat lungs secondary to hypobaric hypoxia lacked smooth muscle α-actin immunoreactivity. [3,11] Work from our lab revealed that the intermediate filament protein nestin was highly expressed in neonatal rat ventricular fibroblasts, significantly downregulated in adult rat ventricular fibroblasts and recapitulated in scar myofibroblasts during the reparative fibrotic response following myocardial infarction. [6] Nestin was also detected in mesenchymal cells identified in the fibrotic lung and kidney and the accumulation of interstitial collagen following injury of the rat and human kidney positively correlated with the density of nestin (+) -interstitial cells.…”

“…[11] Cryosections were stained with the mouse monoclonal anti-nestin (1:400; EMD Millipore), rabbit polyclonal anti-smooth muscle α-actin (1:400; Abcam), rabbit polyclonal anti-collagen type I (1:400; Abcam), or goat monoclonal anti-CD31 (1:100; Santa Cruz Biotechnologies). [14] Adult and neonatal ventricular fibroblasts, rat microvascular and human coronary artery endothelial cells were plated on glass coverslips, fixed with 4% paraformaldehyde and stained with the mouse monoclonal anti-nestin (1:500; EMD Millipore), mouse monoclonal anti-human nestin (1:100; Santa Cruz Biotechnologies), mouse monoclonal anti-eNOS (1:50; BD Bioscience, Mississauga, ON), goat monoclonal anti-CD31 (1:100; Santa Cruz Biotechnologies, Santa Cruz, CA), rabbit polyclonal anti-prolyl 4-hydroxylase alpha polypeptide III (~75 KDa;1:100; EnoGene) or rabbit polyclonal anti-collagen type I (1:400; Abcam).…”

“…[9,10] Moreover, smooth muscle α-actin was not detected in the preponderance of collagen type I-expressing mesenchymal cells identified in fibrotic lungs secondary to hypobaric hypoxia and the fibrotic heart following pressure-overload. [3,11] Work from our lab and others have reported that reactive and reparative fibrosis was characterized by the induction of the class VI intermediate filament protein nestin in a subpopulation of mesenchymal cells. [6,7,11–13] It was further revealed that following renal injury, the magnitude of the reactive fibrotic response positively correlated with the density of the nestin (+) -interstitial cells and exposure of renal-derived collagen-expressing fibroblasts to pro-fibrotic peptide growth factors increased nestin protein levels.…”

“…[3,11] Work from our lab and others have reported that reactive and reparative fibrosis was characterized by the induction of the class VI intermediate filament protein nestin in a subpopulation of mesenchymal cells. [6,7,11–13] It was further revealed that following renal injury, the magnitude of the reactive fibrotic response positively correlated with the density of the nestin (+) -interstitial cells and exposure of renal-derived collagen-expressing fibroblasts to pro-fibrotic peptide growth factors increased nestin protein levels. [13] Biologically, several distinct functions were attributed to nestin including cellular proliferation, migration and a pro-survival anti-apoptotic phenotype.…”