Abstract
BackgroundPrimary cultures endothelial cells have been used as models of endothelial related diseases such atherosclerosis. Biological behavior of primary cultures is donor-dependent and data could not be easily reproducible; endothelial cell lines are emerging options, particularly, human dermal microvascular endothelial cells (HMEC-1), that should be validated to substitute primary cultures for the study of HDL functions.MethodsMorphology, size and granularity of cells were assessed by phase contrast microscopy and flow cytometry of HMEC-1. The adhesion molecules, ICAM-1and VCAM-1 after TNF-α stimulation, and endothelial markers CD105 endoglin, as well as HDL receptor SR-BI were determined by flow cytometry. Internalization of HDL protein was demonstrated by confocal microscopy using HDL labeled with Alexa Fluor 488. HUVECs were used as reference to compared the characteristics with HMEC-1.ResultsHMEC-1 and HUVEC had similar morphologies, size and granularity. HMEC-1 expressed endothelial markers as HUVECs, as well as functional SR-B1 receptor since the cell line was able to internalize HDL particles. HMEC-1 effectively increased ICAM-1 and VCAM-1 expression after TNF-α stimulation. HUVECs showed more sensibility to TNF-α stimulus but the range of ICAM-1 and VCAM-1 expression was less homogeneous than in HMEC-1, probably due to biological variation of the former. Finally, the expression of adhesion molecules in HMEC-1 was attenuated by co-incubation with HDL.ConclusionHMEC-1 possess characteristics of endothelial cells, similar to HUVECs, being a cell line suitable to evaluate the functionality of HDL vis-à-vis the endothelium.
Highlights
Primary cultures endothelial cells have been used as models of endothelial related diseases such atherosclerosis
We determined the optimal concentration of transforming necrosis factor alpha (TNF-α) to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), by a dose-response curve (Fig. 2); we measured the response trough dot plots in terms of quantity of both, ICAM-1 and VCAM-1 expressed as double positive cells for human dermal microvascular endothelial cells-1 (HMEC-1) (Fig. 2A) and human umbilical vein endothelial cells (HUVECs) (Fig. 2B)
Concerning HUVECs, the dot plots showed a wider range of TNF-α-induced expression of ICAM-1 and VCAM-1 (Fig. 2B); responses were observed from doses of 0.75 ng/mL of TNF-α but the dose-response effect was not as regular as for HMEC-1
Summary
Primary cultures endothelial cells have been used as models of endothelial related diseases such atherosclerosis. Some endothelial cell lines have been developed as alternative to primary cell culture with advantages in life span and growth requirements [15] An example of these alternatives are HMEC-1 cells, a microvasculature endothelial cell line developed from human foreskins and transformed with a vector designated as pSVT. HMEC-1 cell line retains endothelial phenotypical characteristics like expression of von Willebrand factor, uptake of acetylated-LDL, and expresses several endothelial markers and adhesion molecules [16]. These characteristics suggest that HMEC-1 would be a suitable model to study lipoproteins-endothelium interactions studies, with lipoproteins, an approach that has not been explored yet. In the present study we analyzed the feasibility of using HMEC-1 cell line as alternative for the study of some HDL properties vis-à-vis the endothelial cells, i.e. regulation of adhesion molecules and HDL internalization
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