Intimal thickening involves transdifferentiation of embryonic endothelial cells

Abstract

Morphological studies have hypothesized different origins for the precursors of the vascular smooth muscle cells (SMCs). The intriguing possibility that intimal SMCs may arise from the endothelium has newly emerged. As a first step towards understanding of the possible mechanisms involved in the transdifferentiation of endothelium into smooth muscle cells, we characterized the in vivo phenotype of the cells located in the aortic wall (distal to the aortic arches). This was accomplished using advanced stages of chicken embryo development. Furthermore, we investigated whether the cells present at the intimal thickening derive from the endothelial cell transdifferentiation. Immunolabeling of serial cryosections suggested that mesenchymal cells observed in the intimal thickening may arise from the endothelium. These cells may persist either as non-muscle throughout the development or possibly convert to cells expressing smooth muscle alpha-actin (SM alpha-actin). To determine whether endothelial cells may actually transdifferentiate into mesenchymal cells, aortic explants from 14-day-old chicken embryos (stage 40) were used. We found that explanted endothelial cells lose their cobblestone-appearance and migrate toward cell-free area. Some of these cells maintain the vWf immunoreactivity, whereas other cells coordinately lose vWf and gain SM alpha-actin expression (transitional cells). Taken together these findings strongly support the possibility that embryonic aortic endothelial transdifferentiate into mesenchymal cells, some of which express SM alpha-actin. Since TGFbeta-3 is considered an essential factor during epithelial to mesenchymal transitions in earlier chicken heart development, we also investigated the distribution of this growth factor at day 14. Our observations indicated that the immunoreactivity for TGFbeta-3 in this stage may be associated with migrating mesenchymal cells and that this immunoreactivity appears to decrease as cell differentiation advances. Therefore, the present study provides evidence that could help to explain 1) the presence of cells displaying a phenotype reminiscent of fetal-like cells in the normal chicken aorta and in the intimal region of the human aorta; 2) the SM lineage diversity in the chicken embryo reported by others; 3) a subpopulation of immature cells in the subendothelial region of the main pulmonary arteries of fetal, neonatal and adult bovines; and 4) the presence of intimal cushions, intimal pads, eccentric and diffuse intimal thickening that are observed in mammalian and avian vessels at birth.

Morphological Changes in the Chicken Ductus Arteriosi During Closure at Hatching

“…Reorientation and migration of smooth muscle cells during this period of hatching correspond with similar events during mammalian DA closure (Yoder et al, 1978. It is possible that the appearance of smooth muscle actin positive cells in the neointima are the result of differentiation of endothelial cells into smooth muscle cells as observed in the developing chicken aorta (Arciniegas et al, 2000).…”

“…Endogenous peroxidase activity was blocked with DAKO dual endogenous enzyme block (S2003) for 10 min followed by protolytic digest for 5 min (DAKO protylitic enzyme, S3007). Sections were incubated with a polyclonal rabbit anit-human von Willebrand Factor primary antibody (A0082, DAKO; 1:100 dilution; Arciniegas et al, 2000) for endothelial cell detection or a monoclonal mouse anti-human asmooth muscle actin primary antibody (clone 1A4, DAKO; Arciniegas et al, 2000). Sections were incubated with the primary antibody for 30 min at room temperature, washed in buffer, and incubated in the secondary antibody (DAKO Envision 1 Dual Link HRP, K4061) for 30 min at room temperature.…”

Integrin-Mediated Endothelial Cell Adhesion and Activation of c-Src, EGFR and ErbB2 are Required for Endothelial-Mesenchymal Transition

“…In view of the above in vitro findings, we evaluated EGF, TGF-, EGFR and ErbB2/Neu by immunoperoxidase staining in the aortic wall at days E12-E14 (stages 38-40) of development when intimal thickening is apparent and EndoMT occurs (Arciniegas et al, 2000). At days E12-E14, the aortic wall is composed by the endothelium, which limits the vessel lumen, and radially oriented mesenchymal cells originating from the endothelium that constitute the intimal thickening.…”

“…Moreover, expression of EGF, TGF-and heparin binding-EGF (HB-EGF) as well as their respective receptors EGFR and ErbB2, have been detected in the intimal thickening and medial smooth muscle cells (SMCs) of atherosclerotic lesions, whereas little or no presence of these molecules is observed in the cells of healthy vessels (Dreux et al, 2006). EndoMT is not only recognized as a phenomenon that occurs during cardiac fibrosis and intimal thickening formation observed in atherosclerosis and restenosis but also in heart and vascular development (Arciniegas et al, 2000;Mironov et al, 2005), pulmonary arterial hypertension (Arciniegas et al, 2007;Morrell et al, 2009;Sakao et al, 2010), cardiac (Goumans et al, 2008) and kidney fibrosis , hyperthrophic scarring (XiQiao et al, 2009), diabetic nephropathy (Li & Bertram, 2010), and during cancer progression . However, there are no studies about the specific role of EGF signalling pathway in the EndoMT process.…”

Possible role of NFĸB in the embryonic vascular remodeling and the endothelial-mesenchymal transition process

“…12 It may involve several regulated steps: cell spreading, cell separation or loss of cell-cell contacts or adherence junctions, proteases, cytokine and growth factor synthesis and secretion, ECM remodeling, membrane receptor expression, cytoskeletal organization, cell separation from the substratum (cell detachment) and cell migration and differentiation. 12 Of particular significance, intimal thickening formation involving EndoMT has been described in both aortic 13 and pulmonary artery wall 14 during advanced stages of chicken embryo development and correlated with an increase in blood pressure that occurs during these stages. Nevertheless, the mechanisms by which endothelial transformation occurs, as well as the signals controlling this process, are only beginning to be elucidated.…”