Spontaneous endothelial-to-mesenchymal transition in human primary umbilical vein endothelial cells

Highlights. Spontaneous endothelial-to-mesenchymal transition of primary human umbilical vein endothelial cells (HUVEC) is characterized by an acquired expression of SNAI2 and TWIST1 genes, loss of endothelial markers and transcription factors (CD31/PECAM1, VE-cadherin, and ERG transcription factor), pronounced expression of S100A4 and ACTA2 genes, and active production of type I collagen, a major component of the extracellular matrix.An optimal algorithm to detect endothelial-to-mesenchymal transition includes gene expression profiling of endothelial lineage markers (PECAM1, CDH5, VWF, ERG), SNAI2 and TWIST1 transcription factors, mesenchymal specification markers (FAP, S100A4, ACTA2) and markers of extracellular matrix synthesis (COL1A1, COL1A2) along with the subsequent negative staining for CD31/PECAM1, VE-cadherin, or ERG and positive staining for intracellular type I collagen.Aim. To  develop  an  algorithm  and  tools  to  determine  endothelial-to-mesenchymal transition (EndoMT) in vitro.Methods. We examined two batches of human umbilical vein endothelial cells (HUVEC) where the first cell batch had a conventional endothelial morphology and the second cell batch underwent a spontaneous EndoMT. Human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) were used as the negative control for EndoMT. Molecular profile was assessed by means of reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining with the further confocal microscopy.Results. In contrast to HUVEC with the physiological profile and arterial ECs, HUVEC undergoing EndoMT lost the expression of endothelial lineage markers (PECAM1, CDH5, VWF, ERG) and acquired the expression of EndoMT transcription factors (SNAI2, TWIST1), mesenchymal markers (FAP, S100A4, ACTA2), and extracellular matrix components (COL1A1, COL1A2) while retaining expression of the common vascular  markers  (HES1,  NRP1).  Western  blotting  analysis  confirmed  the loss of endothelial markers (CD31/PECAM1, VE-cadherin/CDH5, ERG) and demonstrated retained expression of abovementioned vascular markers. Negligible expression of MYH11 and SMTN genes encoding specific contractile markers (smooth muscle myosin heavy chain and smoothelin) in combination with the acquired expression of ACTA2 gene encoding less specific contractile marker alpha smooth muscle actin indicated the phenotypic identity of EndoMT-transformed HUVEC to myofibroblasts but not contractile vascular smooth muscle cells. Loss of immunofluorescence staining of endothelial markers (CD31/PECAM-1, VE-cadherin, and ERG transcription factor) and pronounced intracellular staining of type I collagen testified to the ongoing EndoMT.Conclusion. An  algorithm  to  assess  EndoMT  implies  measurement  of  the  expression  of PECAM1, CDH5, VWF, ERG, SNAI2, TWIST1, FAP, S100A4, ACTA2, COL1A1, and COL1A2 genes in combination with the respective immunofluorescence staining for CD31/PECAM-1, VE-cadherin, or ERG transcription factor and type I collagen.