Proteomic studies on the non-cellular fractions of ECs are so far very limited [21–23], and data on male and female endothelial secretome have not yet been available. In this study, we performed for the first time a proteomic analysis of the secretome from human serum-deprived male and female ECs, showing a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Consistently, a significantly higher percentage of male ECs underwent apoptosis in comparison to female ECs, when exposed to serum starvation as environmental stress. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The knockdown of PTX3 expression revealed its requirement for the proper execution of efferocytosis – that is, the final step of apoptosis in which damaged cells are recruited and removed by macrophages – only in male ECs, but not in female ECs. Taken together, these data suggest a novel and sex-related role for secreted PTX3 in the pathophysiology of human ECs.
Notably, among the differentially abundant secreted proteins, biglycan (BGN) is the only protein encoded by a gene located in sexual chromosomes, specifically in the X chromosome. BGN is a small leucine‐rich proteoglycan acting in its soluble form as an extracellular matrix‐derived danger signal. It has recently been shown that BGN deficiency in the male BALB/cA mouse strain led to sudden death due to aortic rupture, suggesting the potential role of BGN in the maintenance of the structural integrity of the aortic wall. Furthermore, BGN is reduced in individuals with Turner syndrome - a condition that affects only females, where one of the X chromosomes is missing or partially missing - who suffer for vascular anomalies including aortic dissection and rupture .
A sex disparity in the response to stress has been reported in different species and cell types [42–46], and the tendency of male ECs to easier undergo apoptosis in response to serum starvation fully agrees with these results. Overall, it has been suggested that male and female cells adopt different strategies to face a cellular stress induced by the same injury, with male cells more prone to apoptosis, and female cells predisposed to autophagy [42–46]. The different responses to cellular stress, as well as most of the phenotypic differences between male and female cells and organisms, have been related to the sex-biased expression of genes due to their transcriptional and post-transcriptional regulation [47–49]. In humans and other mammals, microRNAs (miRNAs) are key players in the post-transcriptional repression of mRNA targets, and evidence is accumulating for a sex-biased expression of these small regulatory RNAs . To date, few miRNAs have been reported to be present in the Y chromosome whereas the X chromosome contains about 10% of the total miRNAs. Recently, the sex-dependent expression of the miR548am-5p has been proposed to control the propensity of human male dermal fibroblasts to apoptosis through its ability to alter the relative expression of pro- and anti-apoptotic proteins , thus confirming the possible involvement of miRNAs in the regulation of sex-relative properties. In addition, the ability of female ECs to maintain higher levels of metabolites and a better energy balance under stressed conditions in comparison to male ECs has recently been described . Accordingly, the secretome from stressed female ECs contains greater amounts of proteins involved in specific metabolic pathways. Since a central role for metabolism in the phenotypic plasticity characterizing ECs has emerged in recent years , the recruitment of sex-specific metabolic pathways in the cellular response to stress cannot be excluded and deserves further studies.
However, although the regulation of transcription is crucial in defining the specific expression of genes in cells and tissues, it has been shown that most of the tissue-enriched transcriptome codes for secretory proteins - classified as proteins having a signal peptide, but lacking a trans-membrane region - and that secretome holds the largest fraction of tissue-specific proteome . In humans, about 2,600 genes - corresponding to approximately 13% of all protein-coding genes - code for potentially secreted proteins, and around 500 of these proteins were annotated as secreted in the proximity to the cell of origin, including proteins expressed in male/female tissues . Thus, it is possible to hypothesize that specific regulatory programs exert a fine-tune control on the delivery of functional secretory proteins that in turn may be involved in the onset and maintenance of sex-specific cellular properties. Our results, showing different secretory phenotypes in serum-deprived male and female ECs, support the idea that the production of different sets of proteins might take part in the endothelial sex-biased response to cellular stress.
Very recently, it has been discovered that metabolites in the secretome of apoptotic cells are endowed with multiple biological functions, and not simply derived from the passive emptying of dying cells . Some of these metabolites may modulate inflammation and wound healing by inducing specific gene programs in healthy neighbouring cells. Other metabolites may be involved as find-me or eat-me signals in the resolution of apoptosis under which damaged cells are recruited and removed by macrophages via efferocytosis. In this scenario, our finding of elevated PTX3 levels in the secretome of male apoptotic ECs is suggestive of a still unknown role for secreted PTX3 in the establishment of innate sex-dependent properties, e.g., the response to environmental stress in human ECs. Importantly, the increased secretion of PTX3 is closely associated with the apoptotic male phenotype since PTX3 is equally expressed in male and female ECs, and no difference in the secreted quantity has been observed when ECs are not exposed to cellular stressors. About that, it is critical to remark that PTX3 is a cognate molecule of the C-reactive protein (CRP), a prototypic humoral acute phase protein helpful in the clinic as a systemic biomarker to monitor infections and inflammatory diseases [24,37]. Similarly, PTX3 blood levels rapidly rise either in human or mice in pathological conditions with inflammatory and/or infectious origins, and typically increase faster than CRP, very likely because of its local production. Indeed, the human PTX3 protein - a 381 amino acid glycoprotein with a 17-amino acid signal peptide for secretion – can be locally produced by various cell types, including vascular ECs, in response to pro-inflammatory cytokines or microbial moieties, thus acting as a fluid phase pattern recognition receptor able to sense cell and tissue damage and to orchestrate the outcome of the inflammatory response . Hence, it is conceivable that the baseline secretion of PTX3 does not differ between unstressed ECs to precisely increase in response to cellular suffering, e.g. serum deprivation in our in vitro model. In addition, PTX3 has been involved in the regulation of vascular integrity and cardiovascular biology, although contrasting results have been so far provided either in preclinical or clinical research [54,55,35]. Finally, the ability to regulate recognition and clearance of apoptotic cells and debris, namely a role as eat-me signal, has been proposed for PTX3 in different cells and tissues [56,40,57–59]. Our findings, showing that the silencing of PTX3 impaired efferocytosis only in male ECs, but not in female, suggest that this protein might act as an endothelial eat-me-signal in a sex-dependent manner. At variance with other forms of cell death, apoptosis is a non-inflammatory process, and the timely phagocytosis of dying cells by macrophages prevents the release of inflammatory factors, the establishing of inflammation, and the development of chronic inflammatory disorders, such as atherosclerosis [38,39,60]. Therefore, the involvement of PTX3 in the resolution of the male apoptotic process may reflect the effort of secreted PTX3 to maintain vascular integrity and to counteract chronic endothelial inflammation via the prompt execution of the efferocytotic process.
Besides PTX3, our study demonstrated that other molecules with anti-inflammatory properties are present in the male EC secretome. Specifically, it also contains significant levels of calreticulin, one of the most characterized eat-me signals [61,62]. In accordance with our data in PTX3-silenced male ECs, decreased levels of calreticulin have been associated with an impaired efferocytosis . Another noteworthy component of apoptotic male EC secretome is annexin I, which has been actively involved in efferocytosis, in the resolution of inflammation, and in the delay of atherosclerotic plaque progression [64–66]. Moreover, it cannot be excluded that these or other secreted molecules may be responsible for further modulatory actions in nearby cells due to transcriptional/post-transcriptional mechanisms and/or metabolic reprogramming.
At variance with apoptotic male ECs, female EC secretome contains lower quantities of PTX3, calreticulin, and annexin I. In addition, PTX3 silencing left unaffected efferocytosis in female ECs, confirming that the increased secretion of the protein, and its putative role as eat-me-signal, is closely associated with the apoptotic male phenotype. However, these results did not exclude the capability of female ECs to contrast inflammation but perhaps suggest that female cells may temper endothelial inflammation through other mechanisms. As discussed above, male and female cells adopt distinct plans in response to the same cellular stressor [42–46]. Specifically, female cells appear more prone to autophagy, and our preliminary results in serum-deprived female ECs support this hypothesis (Cattaneo et al, manuscript in preparation). Since a protective role against atherosclerosis has been suggested for autophagy , it will be of great interest to study whether this mechanism might represent the path chosen by female ECs to contrast endothelial injures and inflammation.
Perspective and significance
In sum, the finding of different secretory phenotypes in stressed male and female ECs advise a central role for secretory pathways and secreted proteins in the control of sex-specific cellular properties and homeostasis, thus unveiling a novel mechanism that may be responsible for sex-biased pathophysiological responses. We identified secreted PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger, especially in the execution of efferocytosis. Although still debated, the pleiotropic functions of PTX3 in cardio-vascular biology are of growing interest. Our results suggest that a fine tuning in time and space might be responsible for the sex-dependent production/activity of PTX3 in response to environmental stressors. Overall, understanding in what manner PTX3 secretion is regulated in male and female ECs, and in what way it may balance pro- and anti-inflammatory signals at the vascular bed, might be crucial to identify novel sex-specific pathogenetic mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.
Limitations of the study
The main limitation of our study is the use of ECs from a unique source, the human umbilical vein. Actually, in recent years, organ-specificity and function of the endothelium and ECs have been clearly demonstrated . From this perspective, HUVECs derive from a rather unique tissue with distinctive properties (it is, among others, the first interface fetus/mother). Despite this unicity, HUVECs still represent the main model for studying in vitro properties of endothelium and ECs . In fact, HUVECs undergo in vitro angiogenesis, produce NO, and respond to shear stress, hyperglycemia, and inflammatory stimuli, thus recapitulating human disease pathophysiology. In addition, sex-dependent differences in gene expression and functional properties have been shown in male and female HUVECs by us and other groups [12–14,17,18,70], indicating HUVECs as a suitable model for studying cellular biological sex and increasing the translational value of basic research. Nevertheless, whether and through which mechanisms HUVEC dimorphic properties are lifelong conserved and regulated in adult male and female ECs still need to be deeper investigated. However, a transcriptome analysis comparing HUVECs to adult human ECs has very recently shown that more than 250 genes are concordantly differentially expressed between sexes at birth and in adults , thus further supporting the use of HUVECs as a valuable model for basic research on the endothelium.
In addition, in our past and present studies involving HUVECs, we prepared cells from anonymized umbilical cords collected at one of the biggest Obstetrics and Gynaecology public hospital in Milano. Cords were from healthy male or female babies born at term from healthy mothers who were drug- and infection-free. Thus, our cells offer a snapshot of the whole mother/new-born population (without any bias due to stratification or selection). In our opinion, this method of cord collection limits the enrichment of specific phenotypes and make our data even more robust. However, it should be interesting in the future to stratify mother population - by age or cardiovascular risk, for example - to detect whether and how these factors may affect HUVEC response to stressors and/or secretome composition.
Another crucial issue regards the possibility of comparing male and female ECs obtained from dizygotic twin cords to limit genetic and environmental differences. We have already taken advantage of this approach in our previous paper to confirm the increased eNOS expression in female ECs . However, the collection of twin cords is limited by a number of criticisms: i) the incidence of spontaneous dizygotic twinning is quite low, around 1-2%; ii) the rate of preterm labour among twins is around 60%; iii) remarkably, HUVECs from premature cords show impaired properties when compared to cells from term cords ; iv) intrauterine growth restriction is increased in twin pregnancies ; v) about 75% of twins being delivered by cesarean ; vi) when childbirth is vaginal, a correlation between twin-to-twin delivery intervals and pH in umbilical arterial blood gas has been found, thus alerting about metabolic acidosis . In addition, it should be considered that some differences may exist between the prenatal environment of male and female dizygotic twins. Specifically, exposure to androgens, notably testosterone, may be particularly high for the female twin in opposite sex twin pairs, also in comparison to female in monozygotic twins or unrelated new-borns. Studies in rodents have shown that chromosomal female foetuses that are in proximity to male littermates are passively exposed to testosterone and exhibit masculinized morphological, endocrine, and behavioural phenotypes . Similarly, human females exposed prenatally to a male co-twin experience in utero testosterone transfer that induces cognitive and behavioural changes . Indeed, in the human embryo, testosterone directly impacts on developing brain structures and starts to be produced in male testes 6 weeks after conception . The ability of testosterone to de-feminize and masculinize gene expression in a XX background is mediated by epigenetic mechanisms, and finally influences gene expression . In general, studies in twins help to discover and/or amplify less pronounced sex differences due to the greatest statistical power of the paired design. At variance, sex differences identified in unrelated new-borns may be robust and prevalent.