Sex-dependent differences in the secretome of human endothelial cells

Abstract

Background: Cellular sex has been rarely considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and ‘omics approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms.Methods: We performed proteomic and gene ontology (GO) analyses of secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and western blot techniques, and efferocytosis through the ability of the macrophage cell line RAW-264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. Results: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison to female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggests that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seems to be independent on PTX3 expression. Conclusions: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

Serum starvation induces sexual dimorphisms in secreted proteins of human umbilical vein endothelial cells (HUVECs) from twin pairs

“…Very recently, sex-specific differences in secreted proteins in male and female HUVEC of nonrelated pregnancies after overnight serum starvation were analyzed in another study [31]. In contrast to our results, Cattaneo et al found a higher number of starvation-responsive secreted proteins in male cells compared to female HUVECs (20 and 3 proteins in male and female HUVECs, respectively).…”

“…Very recently, sex‐specific differences in secreted proteins in male and female HUVEC of nonrelated pregnancies after overnight serum starvation were analyzed in another study [31]. In contrast to our results, Cattaneo et al.…”

“…The authors observed a significant enrichment of cellular stress proteins and of proteins related to the regulation of apoptosis in the secretome of male HUVECs. Higher levels of the proapoptotic pentraxin-related protein (PTX3) in the medium of male HUVECs contributed to higher apoptosis and phagocytosis in male HUVECs after serum starvation [31]. In our study, we found only nonsignificant 1.1-fold higher levels of PTX3 in the supernatants of male HUVECs at both time points of serum starvation with n = 15 male/female twin pairs.…”

Why you should pay more attention to your cells’ sex

“…Our data are further supported by a comparative analysis of gene expression data from endothelial cells of twin pairs at birth and adult non-twins, which has shown that sex differences exist at all stages of life and that 14-25% of the overall endothelial cell transcriptome is sex-specific (Hartman et al, 2020). Beyond gene dosages, sex differences were likewise identified in secreted proteins of cell culture supernatants as well as in various cellular functions such as proliferation, angiogenesis and migration (Addis et al, 2014;Cattaneo et al, 2017Cattaneo et al, , 2021Lorenz et al, 2019Lorenz et al, , 2022. For example, we and others have found that female HUVECs have better stress tolerance and higher migratory capacity than male cells (Addis et al, 2014;Cattaneo et al, 2021;Lorenz et al, 2015Lorenz et al, , 2019.…”

Remarkable sex-specific differences at Single-Cell Resolution in Neonatal Hyperoxic Lung Injury

“…Significantly, we highlight the sex-specific differences in gene expression in different lung cell sub-populations in the epithelial, endothelial, and immune cells. Differences in the secretome and transcriptome of human umbilical venous endothelial cells has been described (120) and so is the response to hyperoxia in human pulmonary microvascular endothelial cells (121). The immune cells stand out in the degree of sexual dimorphism in the hyperoxia exposed neonatal lung.…”