Phenotypic Sexual Dimorphism of Micro‐ and Macrovascular Endothelium with Respect to Size, Growth, and Wound Repair

Abstract

Endothelial cell biology has identified heterogeneity amongst endothelial cells (EC) of macro‐ and microvascular origin as well as by organ and position in the vascular tree but considerably less is known about the contribution of sex Further the influence of sex on basal endothelial phenotype has not been explored so we have been undertaking studies to test the hypothesis that the endothelial phenotype is independent of sex under basal conditions. In this study we concentrated on the traits of cell size, morphology following growth to confluence on collagen, growth rate following plating and time to repair following wounding. Endothelial cells were isolated from the aorta (macrovessel) and abdominal free wall (skeletal muscle, SKM, microvessel) adult, age‐matched male and female Sprague Dawley rats and selected by binding to CD31‐coated magnetic beads. Cells were plated at the same density on collagen coated plates under identical conditions of culture and studies were performed at passage 8 when confluence was achieved. Verification of sex of the cells was confirmed by the presence of the SRY gene in EC from the males.Genomic sex influenced all the parameters tested in different ways. Free cell size determined with the cellometer revealed that Male Aortic EC = Male SKM EC = Female SKM EC > Female Aortic EC (Median 16.1, 15.6, 16 and 14.1 μm diameter, respectively). Once spread on collagen Male SKM = Female SKM > Female Aorta > Male Aorta (Median 856, 951, 657 and 576 μm2 area, respectively). Following plating at equal density no sex differences were observed with respect to growth for EC of aortic origin or between Aortic and SKM EC of males; those from female SKM, though had only multiplied 5‐fold by day 6 as compared to the 10‐fold increase in number of the other sources. Finally in a series of wound closure assays, where the wounds were of comparable size, no sex differences were observed for the EC of SKM origin with complete closure being observed at 39 and 40 hours (male and female, respectively). By contrast two features were observed in the wound healing response of aortic EC: first there was significant wound retraction 2 hours following injury of the male aortic EC and they took longer to heal (45 vs 38 hours for male and female, respectively).Our data demonstrate that not only do EC differ by origin (macro‐ vs. microvessel) but also by sex and in a variety of features that point to multiple sexually dimorphic mechanisms. Further these data demonstrate the fallacy of assuming a lack of sexual dimorphism under basal conditions but also a lack of a role for sex hormones in the control of these features as well as the implication that multiple cellular mechanisms are responsible for the determination of endothelial cell phenotype of males and females respectively.Future work will focus on the mediators of growth and morphology and associated 2nd messenger pathways under basal and conditions show at the whole vessel level to alter function in a sexually dimorphic manner.Support or Funding InformationSupported by NIH R01 DK095501 and Pulmonary Medicine Research Fund