Sex-dependent differences in the secretome of human endothelial cells

Background Cellular sex has rarely been 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 the 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 with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed 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. Supplementary Information The online version contains supplementary material available at 10.1186/s13293-020-00350-3.


Background
Almost all of our knowledge about fundamental biological processes has been provided by primary and established cell lines. However, most of these studies disregarded the sex origin of cells, and sex has been rarely considered as a relevant biological variable in preclinical research [1,2]. This asexual approach has limited the understanding of potential sex-based differences not only in basic biological functions but especially in pathophysiological mechanisms. This loss of knowledge is particularly restrictive when the pathogenesis of diseases showing predictable sex differences is studied [3,4].
Atherosclerosis and cardiovascular disease (CVD) are classical examples of diseases where sex/gender differences have been described [5]. A signi cant body of evidence suggests that CVD is less prevalent in women than men until midlife, and the female advantage in younger women has been attributed to estrogens, which are lost with menopause. Since the earliest event in the onset of atherosclerosis and CVD is endothelial dysfunction, many studies have been focused on endothelium and endothelial cells (ECs). Although evidence of important sex/gender differences in the endothelial function has been reported in both rodents and humans [6,7], the sex of cells was not consistently reported in studies involving ECs, even when the effects of sex hormones were analyzed [8][9][10]. However, when primary cultures of male and female ECs from different vascular beds were independently studied, some inborn sex differences appeared [11][12][13][14][15][16][17][18].
In this context, proteomics, and 'omics approaches in general, can provide powerful tools to obtain detailed cellular maps at the molecular level, thus favoring the comprehension of the molecular basis of pathogenesis. Sex/gender speci c differences at the proteomic level throughout non-sexual organs and tissues have been described [19]. Such differences have been reported also for dioichous plants and for many animals in all phyla [19]. However, molecular data for human tissues and primary cells are still scarce, as well as proteomic studies focused on sex-related differences in ECs [17,20]. Likewise, very few proteomics studies have analyzed the EC secretome [21][22][23], that consists of all the proteins released by vascular cells, and that controls a plethora of biological processes, thus representing a potentially source for biomarkers and therapeutic target discoveries.
In this study, we have performed for the rst time a proteomic analysis of the secretome from human male and female ECs isolated from umbilical cords (Human Umbilical Vein ECs, HUVECs). Among others, we consistently found higher levels of the long Pentraxin 3 (PTX3), a highly conserved member of the pentraxin family [24], in the secretome of male ECs. Therefore, we focused our attention on the biological meaning of the observed sex-dimorphic production of PTX3, by evaluating its role in the response of ECs to an external source of cellular stress represented by serum deprivation.

Cell cultures
HUVECs were freshly isolated from umbilical cords essentially as previously described [25]. Cells were pooled from two or more donors -to minimize variability associated with cells derived from a single male or female newborn donor -and used at passages 1-5. HUVECs were routinely grown in 199 medium supplemented with 20% fetal bovine serum (FBS), 25 µg/ml endothelial cell growth supplement (ECGS), and 50 mg/ml heparin on 0.1% gelatin-coated surfaces. All the experiments were performed on ECs or conditioned media collected after an overnight incubation in the absence of serum or in the presence of 2% FBS, unless otherwise indicated.
Label-free mass spectrometry (LC-MS E ) analysis Secretome samples for proteomic analysis were desalted, concentrated and digested as previously described [23]. After lyophilization, the secreted protein pellets were dissolved in 25 mmol/L NH 4 HCO 3 containing 0.1% RapiGest (Waters Corporation), sonicated, and centrifuged at 13,000g for 10 min. Samples were then incubated 15 min at 80°C and reduced with 5 mmol/L DTT at 60°C for 15 min, followed by carbamidomethylation with 10 mmol/L iodoacetamide for 30 min at room temperature in the darkness. Then, sequencing grade trypsin (Promega) was added to each sample (1 mg every 50 mg of proteins) and incubated overnight at 37°C. After digestion, 2% TFA was added to hydrolyze RapiGest and inactivate trypsin. Tryptic peptides were used for label-free mass spectrometry analysis, LC-MS E , performed on a hybrid quadrupole-time of ight mass spectrometer coupled with a nanoUPLC system and equipped with a Trizaic source (Waters Corporation) as previously described in details [23,26]. Statistical analysis has been performed by means of Progenesis QIP v 4.1 (Nonlinear Dynamics) using a Uniprot human protein sequence database (v2017-1). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [27] partner repository with the dataset identi er PXD020375 and 10.6019/PXD020375.

Gene Ontology analysis
Data were analyzed with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 11.0) database [28] as previously described [29] to identify enriched gene ontology (GO) terms in the biological process, molecular function or cellular component categories. We speci cally employed the enrichment widget of STRING, which calculates an enrichment P-value based on the Hypergeometric test and the method of Benjamini and Hochberg for correction of multiple testing (P value cut-off of <0.05).

Measurement of secreted PTX3
Conditioned media (2.0-2.5 ml/ ask) were collected after an overnight incubation from con uent HUVEC monolayers cultured in 25 cm 2 -gelatin coated asks. After a 10-min centrifugation (300g at 4°C) to remove cellular debris, media were aliquoted and stored at -80°C. The concentration of PTX3 was measured using the Human Pentraxin 3/TSG-14 Immunoassay (R&D Systems Inc).

Cell images
Photographs of HUVECs were acquired at a 10x magni cation with an Olympus U-CMAD3 phase contrast microscope equipped with an Olympus digital camera.

Measurement of adherent cells
The number of adherent cells was measured by crystal violet on HUVECs plated at a density of 2.0x10 4 cells/well in 0.1% gelatin-coated 96-well microplates the day before the experiment. Crystal violet binds to DNA and proteins, thus allowing the detection of adherent cells [30]. Brie y, cells were xed with ice-cold 100% methanol for 10 min and stained with crystal violet (0.5% in 20% methanol) for about 15-20 min. After multiple washes with deionized water, the plate was air-dry, and crystal violet stain was solubilized in DMSO (100 µl/well) before the measurement of optical density at 595 nm by a multiplate reader (Victor™, PerkinElmer).

Measurement of Reactive Oxygen Species (ROS)
ROS were detected as previously described [31] on HUVECs plated at a density of 2.0x10 4 cells/well in 0.1% gelatin-coated black 96-well microplates the day before the experiment. Cells were loaded for 30 min at 37°C in the dark with the uorescent dye 5(6)-Carboxy-2′7′-dichloro uorescein diacetate (CM-DCFDA, 10 µM) in HBSS buffer (Hepes 25 mM pH 7.4, NaCl 120 mM, KCl 5.4 mM, CaCl 2 1.8 mM, NaHCO 3 25 mM, glucose 15 mM) containing 1% FBS. Afterwards, the cells were washed, and uorescence was assessed after 30 min by a multiplate reader (Victor™, PerkinElmer) with excitation and emission wavelengths of 485 and 530 nm, respectively. The protein content of each well was quanti ed by the BCA assay (Pierce) to normalize sample-to-sample variation.

Apoptosis assays
Quanti cation of apoptosis/necrosis was performed by Annexin V-FITC conjugate and propidium iodide (PI) staining (Immunological Sciences) followed by uorescence activated cell sorting (FACS) performed with a FACScalibur ow cytometer equipped with a 488 nm argon laser (Becton Dickinson). The collected data were evaluated by Cell Quest software. In addition, active caspase-3 and PARP-1 proteins were detected by conventional western blot on total cell lysates prepared in SDS-PAGE sample buffer (62 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.04% bromophenol blue). Densitometric analyses of immunoblots were performed using the National Institute of Health (NIH) Image J software package. Full length unedited blots are shown in the Additional le 2.

Small interfering RNA (siRNA) transfection
To silence PTX3 expression, HUVECs were transfected with the Hs_PTX3_1 FlexiTube siRNA duplexes against human PTX3 (SI00695947, Qiagen). An AllStars Negative Control FlexiTube siRNA (Qiagen) was used as control. Both siRNAs were individually transfected at a 5 nM concentration using the PepMute transfection reagent according to the manufacturer's instructions (Signa Gen Laboratories). For in vitro efferocytosis assays, cells were transfected 48h before calcein-AM loading and overnight serumdeprivation. The knockdown of PTX3 expression was analyzed by reverse transcription and quantitative real time PCR (RT-qPCR).
Efferocytosis assay RAW 264.7 macrophages were plated at a density of 5.0×10 4 cells/well in 100 µl/well of DMEM + 5% FBS in a black 96-well microplate. HUVECs were loaded for 30 min with calcein-AM (2 µM) in HBSS before overnight incubation in 2% FBS-containing medium. After detachment and re-suspension in the conditioned media, HUVECs were collected by centrifugation and overlaid on the RAW 264.7 cells at a 1:1 ratio of macrophages:HUVECs in a 100 µl volume of 20% FBS-containing 199 medium [33]. A 100 µl aliquot of HUVEC suspension was concurrently plated in empty wells to measure the total uorescence added to macrophages. Following 2h of incubation, cells were washed 3 times with HBBS -to remove cells that were not engulfed -and the Raw 264.7-associated uorescence was assessed by a multiplate reader (Victor™, Perkin Elmer). Data are expressed as percent (%) of engulfed cells, that is the RAW 264.7 cell-associated uorescence divided by the total uorescence multiplied by 100.

Statistical procedures
Unless otherwise indicated, data are expressed as mean ± s.e.m. of at least 3 independent experiments performed on different cell preparations. Statistical analysis was carried out using unpaired Student's ttest or 2-way analysis of variance (ANOVA) followed by Sidak's multiple comparison test. In the 2-way ANOVA analyses, we considered as row factor the sex of cells, and as column factor the treatment (20 vs 2% FBS in Figs. 2D and 3A/B, and ctrl vs PTX3 siRNA in Fig. 5C). P-values of <0.05 were considered signi cant. All the analyses were performed using the GraphPad Prism software (version 8.4.2).

Proteomic analysis of male and female EC secretome
To evaluate still unknown differences in terms of protein secretion between human male and female HUVECs (abbreviated as ECs), we analyzed their secretome after an overnight incubation in the absence of serum by means of a mass spectrometry based proteomic approach, LC-MS E . Label free LC-MS E analysis allowed us to identify and quantify EC secreted proteins (dataset PDX020375). Among these proteins, we identi ed 20 proteins signi cantly more abundant in the secretome of male ECs, and 3 proteins more present in the female secretome (Table 1). Of note, a gene ontology (GO) analysis, performed with STRING on the panel of proteins increased in the male secretome, showed a signi cant enrichment in the biological process categories of GO terms related to responses to stress (p = 0.0029) and to cytokine stimulus (p = 0.01), and to the regulation of apoptosis (p = 0.014) ( Fig. 1 and Supplementary table 1, see Additional le 1). These results suggest that male and female ECs show different secretory phenotypes, due the secretion of different sets of proteins, in response to serum deprivation. Among the proteins differentially secreted by male and female ECs, we focused our interest on PTX3 for two main reasons: i) a role for PTX3 has been proposed in the response to vascular damage and in the development and progression of atherosclerosis [34,35]; ii) we and others have previously shown that PTX3 is one of the more represented protein in the secretome of human ECs when cells are studied without sex-segregation [21][22][23]. Despite these suggestions, the biological signi cance of the secreted PTX3 in the human EC pathophysiology is still unknown as well as the existence of a possible sex dimorphism in its production and/or biological role. The presence of PTX3 in the conditioned media obtained from male or female ECs incubated overnight in serum-free or in 2% FBS-containing media was validated by a quantitative immune-enzymatic assay. As shown in Fig. 2, the levels of PTX3 were signi cantly higher in male ECs in comparison to female ECs either in the absence of serum ( Fig. 2A, 8.1 ± 0.4 vs 5.3 ± 0.1 ng/ml for male and female ECs, respectively) or in the presence of 2% FBS (Fig. 2B, 10.6 ± 1.2 vs 4.5 ± 1.5 ng/ml for male and female ECs, respectively). The difference in the levels of secreted PTX3 was still conserved when male/female ECs were incubated overnight in 0.5% bovine serum albumin-containing medium (data not shown). At variance, no signi cant differences between male and female ECs were observed when PTX3 was measured in media from cells incubated overnight in standard conditions, i.e. 20% FBS (Fig. 2C). Notably, when control and serum-deprived conditioned media were compared, the amount of secreted PTX3 was increased only in male ECs (Fig. 2D). No signi cant differences were observed between control and starved female ECs, where a tendency to a decrease rather than an increase in the PTX3 levels was observed (Fig. 2D). Finally, the constitutive expression of PTX3 measured by RT-qPCR did not differ between male and female ECs (Fig. 2E). Collectively, these results reveal different amounts of PTX3 in the secretome of starved male and female ECs, thus suggesting a possible sex-dependent function for secreted PTX3 in the endothelial response to serum deprivation.
Serum starvation acts as a stressor in male and female ECs Serum starvation represents an important stress factor, that deprives cells of crucial metabolites and growth factors, thus breaking the physiological cellular homeostasis. Consequently, cells can undergo morphological alterations, accumulate reactive oxygen species (ROS), stop to growth, and eventually die.
After an overnight incubation in 2% FBS-containing medium: i) the number of adherent ECs, evaluated by crystal violet staining, was signi cantly reduced in comparison to control cells, i.e. cells incubated in 20% FBS, in both sexes (by 20.7 ± 4.2 and 12.7 ± 3.0% for male and female ECs, respectively; p = 0.152, n = 6) (Fig. 3A); ii) the intracellular ROS content, measured with the uorescent dye DCFDA, was increased by about 2-fold in both male and female ECs (Fig. 3B). Accordingly to our previous results, no signi cant differences in intracellular ROS and cell number were found between male and female ECs cultured in 20% FBS [14,36]. Of note, phase contrast microscopy images of male and female EC monolayers incubated overnight in 2% FBS showed the occurrence of cellular debris, especially in male ECs (Fig. 3C). These results demonstrate that an overnight serum starvation adversely affects EC behavior.

Serum Starvation Induces Apoptosis In Male Ecs
Cells can adopt multiple strategies in response to external stressors, such as serum deprivation, ranging from the activation of pathways that promote survival to eliciting programmed cell death to remove damaged cells. In this context, a central role for PTX3 in the response to tissue damage and repair has been proposed [37]. Thus, to shed light on the defense mechanisms activated by ECs in response to serum starvation, and to elucidate potential sex differences in the role of PTX3 in the execution of these strategies, we analyzed the apoptotic process. To this aim, annexin V-conjugated FITC and PI staining followed by FACS analysis were used to detect apoptotic cells in male and female ECs incubated overnight in 2% FBS-containing medium. Starved male ECs showed higher percentages of both early and late apoptotic cells -sorted in the upper left (UL) and in the upper right (UR) quadrants, respectively -in comparison to female ECs (Fig. 4A), with the greater increase in the fraction of cells engaged in the early apoptotic process (30.4 ± 2.0 and 15.0 ± 2.3% for M-ECs and F-ECs, respectively) (Fig. 4B). Collectively, apoptotic cells (expressed as the sum of cells sorted in both the UL and UR quadrants) account for about 50% of the gated cells in male ECs whereas only 25% of the cells was apoptotic in female ECs (Fig. 4B).
No signi cant differences were observed between ECs of both sexes in the percentage of necrotic cells (lower right quadrant, LR) (1.0 ± 0.6 and 0.6 ± 0.4% for M-ECs and F-ECs, respectively). The higher tendency of male ECs to undergo apoptosis was con rmed by western blot analysis of serum-deprived male and female EC lysates. The 17-and 12-kD bands corresponding to the cleaved form of caspase-3one the key executioners of apoptosis -were signi cantly higher (by 3.45 ± 0.91 fold, n = 4) in lysates from starved male ECs in comparison to female ECs (Fig. 4C). Likewise, the 89-kD fragment of the cleaved poly (ADP-ribose) polymerase (PARP-1) -one of the main targets of activated caspase-3 -was evident only in lysates from serum-starved male ECs (Fig. 4D). All these results unveil a different tendency of male and female ECs to undergo apoptosis in response to an external stressor represented by serum starvation.
The expression of PTX3 is required for efferocytosis in male ECs To properly conclude the apoptotic process, damaged cells need to be e ciently removed in vivo by tissue macrophages via efferocytosis [38,39]. This process can be reproduced in vitro, and the number of apoptotic cells engulfed by a macrophage cell line can be quanti ed [33]. When calcein-loaded ECs exposed overnight to 2% FBS were incubated with a monolayer of RAW 264.7 cells, a comparable fraction of male and female ECs was engulfed (37.0 ± 9.7% and 27.2 ± 6.0%, respectively) (Fig. 5A). A role for PTX3 in the clearance of apoptotic neutrophils and in their uptake by macrophages has been proposed [40]. Thus, we silenced PTX3 to reveal its putative involvement in in vitro male and female EC efferocytosis. The ability of siRNA (5 nm for 48 h) to e ciently knock-down PTX3 expression in both M-ECs and F-ECs is shown in Fig. 5B. Outstandingly, the percentage of phagocytized male ECs was decreased by about 35% in silenced cells, whereas the engulfment of female ECs was slightly increased (by about 20%) in the absence of PTX3 expression (Fig. 5C). Therefore, male ECs is dependent on the expression of PTX3 to properly carry out the efferocytotic process. At variance, female EC efferocytosis seems to be independent of PTX3 expression.

Discussion
Proteomic studies on the non-cellular fractions of ECs are so far very limited [21][22][23], and data on male and female endothelial secretome have not yet been available. In this study, we performed for the rst time a proteomic analysis of the secretome from human serum-deprived male and female ECs, showing a signi cant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Consistently, a signi cantly 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 efferocytosisthat is, the nal step of apoptosis in which damaged cells are recruited and removed by macrophagesonly 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.
A sex disparity in the response to stress has been reported in different species and cell types [41][42][43][44][45], 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 [41][42][43][44][45]. 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 sexbiased expression of genes due to their transcriptional and post-transcriptional regulation [46,47]. However, although the regulation of transcription is crucial in de ning the speci c expression of genes in cells and tissues, it has been shown that most of the tissue-enriched transcriptome codes for secretory proteins -classi ed as proteins having a signal peptide, but lacking a trans-membrane region -and that secretome holds the largest fraction of tissue-speci c proteome [48]. In humans, about 2,600 genescorresponding 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 [49]. Thus, it is possible to hypothesize that speci c regulatory programs exert a ne-tune control on the delivery of functional secretory proteins that in turn may be involved in the onset and maintenance of sex-speci c 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 do not simply derived from the passive emptying of dying cells [50]. Some of these metabolites may modulate in ammation and wound healing by inducing speci c gene programs in healthy neighbouring cells. Other metabolites may be involved as nd-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 nding 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 to 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. The human PTX3 protein is a 381 amino acid glycoprotein, including a 17 amino acid signal peptide for secretion, with complex regulatory functions at the crossroads of innate immunity, in ammation, and tissue repair [37,24]. PTX3 can be produced locally by various cell types, including vascular ECs, in response to pro-in ammatory cytokines or bacterial molecules engaging Toll-like receptor. 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 [51,52,35]. Finally, the ability to regulate the "comestible status" of apoptotic cells and debris, namely a role as eat-me signal, has been proposed for PTX3 in different cells and tissues [53,40,[54][55][56]. Our ndings, 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-in ammatory process, and the timely phagocytosis of dying cells by macrophages prevents the release of in ammatory factors, the establishing of in ammation, and the development of chronic in ammatory disorders, such as atherosclerosis [38,39,57]. Therefore, the involvement of PTX3 in the resolution of the male apoptotic process may re ect the effort of secreted PTX3 to maintain vascular integrity and to counteract chronic endothelial in ammation via the prompt execution of the efferocytotic process.
Besides PTX3, our study demonstrated that other molecules with anti-in ammatory properties are present in the male EC secretome. Speci cally, it also contains signi cant levels of calreticulin, one of the most characterized eat-me signals [58,59]. In accordance with our data in PTX3-silenced male ECs, decreased levels of calreticulin have been associated to an impaired efferocytosis [60]. Another noteworthy component of apoptotic male EC secretome is annexin I, that has been actively involved in efferocytosis, in the resolution of in ammation, and in the delay of atherosclerotic plaque progression [61][62][63].
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, con rming that the increased secretion of the protein, and its putative role as eat-me-signal, is closely associated to the apoptotic male phenotype. However, these results did not exclude the capability of female ECs to contrast in ammation but perhaps suggest that female cells may temper endothelial in ammation through other mechanisms. As discussed above, male and female cells adopt distinct plans in response to the same cellular stressor [41][42][43][44][45]. Speci cally, 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 [64], it will be of great interest to study whether this mechanism might represent the path chosen by female ECs to contrast endothelial injures and in ammation.

Conclusions
The nding 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-speci c cellular properties and homeostasis, thus unveiling a novel mechanism that may be responsible for sex-biased Gene ontology analysis of the proteins more secreted by male ECs (see Table 1). String network generated with the proteins more abundant in the male EC secretome highlighting the enriched biological processes: red, cellular response to stress; blue, cellular response to cytokine stimulus; green, regulation of apoptotic process.

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