Sex Influence on Autophagy Markers and miRNAs in Basal and Angiotensin II-Treated Human Umbilical Vein Endothelial Cells

Cardiovascular diseases (CVD) display many sex and gender differences, and endothelial dysfunction, angiotensin II (Ang II), and autophagy represent key factors in the autophagic process Therefore, we studied whether Ang II modulates the mentioned processes in a sex-specific way in HUVECs obtained from healthy male and female newborns. In basal HUVECs, the Parkin gene and protein were higher in FHUVECs than in MHUVECs, while the Beclin-1 protein was more expressed in MHUVECs, and no other significant differences were detected. Ang II significantly increases LAMP-1 and p62 protein expression and decreases the expression of Parkin protein in comparison to basal in MHUVECs. In FHUVECs, Ang II significantly increases the expression of Beclin-1 gene and protein, and Parkin gene. The LC3 II/I ratio and LAMP-1 protein were significantly higher in MHUVECs than in FHUVECs, while Parkin protein was significantly more expressed in Ang II-treated FHUVECs than in male cells. Ang II affects the single miRNA levels: miR-126-3p and miR-133a-3p are downregulated and upregulated in MHUVECs and FHUVECs, respectively. MiR-223 is downregulated in MHUVEC and FHUVECs. Finally, miR-29b-3p and miR-133b are not affected by Ang II. Ang II effects and the relationship between miRNAs and organelles-specific autophagy is sex-dependent in HUVECs. This could lead to a better understanding of the mechanisms underlying sex differences in endothelial dysfunction, providing useful indications for innovative biomarkers and personalized therapeutic approaches.


Introduction
Cardiovascular diseases (CVD) are the primary cause of death worldwide [1].Importantly, there are numerous sex and gender differences in presentation, diagnosis, natural course, treatments, and outcomes of CVD such as hypertension, ischemic heart disease, heart failure, vascular complications of diabetes mellitus, ictus, etc. [2][3][4].Notably, endothelial dysfunction is a hallmark of CVD, and is also a predictor of CVD outcomes [5].It has been reported that microvascular dysfunction may contribute to ventricular remodelling and leads to an increased likelihood of heart failure hospitalizations [6,7].Furthermore, endothelial dysfunction can promote atherosclerosis, increasing morbidity and mortality for severe cardiovascular events [8].This is especially evident in women [9], and some of the sex differences in CVD can be attributed to differences in endothelial dysfunction [10].
The endothelium plays a key role in vascular and immune functions, being involved in blood pressure regulation, angiogenesis, thrombotic state, atherosclerosis and inflammation, etc. [11,12].Endothelial functions are governed by numerous factors such as lifestyle, behavioral factors, gut microbiota, sympathetic and immune activation, sexual and vasoactive hormones such as the renin-angiotensin-aldosterone system (RAAS) [13][14][15][16].Preclinical and clinical studies support the hypothesis that RAAS mainly contributes to the pathogenesis of some CVDs.For example, angiotensin II (Ang II) accelerates hypertension [17] and promotes atherosclerosis [18] and endothelial dysfunction [19].Moreover, it has been reported that Ang II and other components of RAAS can operate in a sex-dependent manner [2,[20][21][22].
A process that contributes to sex differences in the physiology and pathophysiology of CVD is autophagy [23][24][25][26][27]. Physiologically, this cannibalistic process degrades and recycles cellular components providing essential nutrients for cell survival [28], while excessive or defective autophagic activity is not compatible with cellular and organismal homeostasis, and aberrant autophagy is generally associated with a wide range of pathologies [23,24].Autophagy involves three stages: induction of autophagy, nucleation of the autophagosome, and elongation of the autophagosome [28], which are characterized by specific markers.Briefly, Beclin-1 is involved in the formation of the pre-autophagosome, LC3I and its delipidated form LC3II are involved in the autophagosome formation [28], and the lysosomal membrane protein LAMP-1 is involved in autophagosomes fusion with endosomes/lysosomes [28].
Selective forms of autophagy that degrade specific organelles as damaged mitochondria (mitophagy) are also described [28].Mitophagy mainly occurs through ubiquitinmediated mitophagy, which is linked to Parkin, which acts as a downstream protein of PINK1 [29].In the early 1960s, the first sex differences in mitochondria were described, and a multitude of differences have been found, which have been reviewed in detail [30].In some organs and tissues, mitophagy is influenced by sex, but data are not available in HUVECs, although aberrant mitophagy has been related to numerous CVDs including atherosclerosis [29,31,32].Progress has been made in mechanisms governing selective autophagy receptors such as p62.The multifunctional protein adaptor p62 participates in mitophagy and cargo autophagy and has many other activities [29].For example, disturbance of mitophagy is accomplished by an accumulation of p62, which is responsible for bringing polyubiquitinated proteins to the autophagosome [32].
It has recently been discovered that the different steps of the selective and non-selective autophagic process can be finely regulated by micro-RNAs (miRNAs) [33] whose levels, in some cases, may be regulated by sexual hormones [34].Some miRNAs can inhibit the initial formation stage of autophagosomes by inhibiting the expression of Beclin-1, while others inhibit the elongation stage by working directly on LC3 [33].For example, the role of miR-133a in the regulation of mitophagy was demonstrated in mice [35].Therefore, it is emerging that some miRNAs are new players in the regulation of autophagy, and alterations of autophagy-related miRNAs are implicated in several pathological conditions like cancer, neurodegeneration, CVD, and infections [36].Interestingly, autophagy may itself modulate miRNA levels, as they are selectively sequestered and degraded by the lysosomal pathway [33].In addition, miRNAs act on RAAS [37,38], and their levels are modified during hypertension [39,40].The exact role of Ang II in endothelial autophagy has not been fully clarified, although autophagy may play a key role in CVD, including endothelial dysfunction [32,41]; even less is known whether these activities of Ang II are influenced by sex.
It has become crucial to study sex's influence on autophagy in primary HUVECs looking at genes, miRNAs, and proteins either in basal or in Ang II-treated cells.This knowledge will improve our therapeutic approaches in CVD because until now, in the majority of cases, men, male animals, and cells of unknown sex have been used, generating scarce knowledge of female needs in health and diseases.

Characteristics of Donors
The mothers of female and male neonates did not differ significantly in age, body weight at the beginning and the end of the pregnancy, and body mass index.Neonates of both sexes did not diverge significantly in gestational age and body weight at birth (Table 1).

LDH Release and MDA Levels
In basal male and female cells, LDL release was similar, as were MDA levels.In male and female HUVECs (MHUVECs and FHUVECs, respectively), the different doses of Ang II did not significantly change the release of LDH, and MDA levels were not changed by 0.5 µM (Figure S1).

Viability
In basal cells, viability was not statistically significantly different in the two sexes (Figure 1).The dose-response curve of Ang II showed a clear sex effect (Figure 1).In particular, the treatment with Ang II of FHUVECs had no significant effect on viability, although there was a tendency to decrease when compared to basal cells.In MHUVECs, the treatment with Ang II dose-dependently increased the viability (Figure 1).Linear regression analysis showed that slopes significantly diverged between MHUVECs and FHUVECs (Figure 1).

Effect of Ang II on HUVECs Migration
In basal conditions, male and female HUVECs did not present any significant difference in the number of migrated cells (4983 ± 472 cells in FHUVECs and 6630 ± 760 in FHU-VEC, n = 5 for each sex; p = 0.79).In both male and female HUVECs, treatment with Ang II significantly reduced migration to a similarl degree (by about 35% and 40% in MHU-Figure 1. Linear regression analysis of the effect of angiotensin II on the viability of MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.

Effect of Ang II on HUVECs Migration
In basal conditions, male and female HUVECs did not present any significant difference in the number of migrated cells (4983 ± 472 cells in FHUVECs and 6630 ± 760 in FHUVEC, n = 5 for each sex; p = 0.79).In both male and female HUVECs, treatment with Ang II significantly reduced migration to a similarl degree (by about 35% and 40% in MHUVECs and FHUVECs, respectively) (Figure 2).

Effect of Ang II on HUVECs Migration
In basal conditions, male and female HUVECs did not present any significant difference in the number of migrated cells (4983 ± 472 cells in FHUVECs and 6630 ± 760 in FHU-VEC, n = 5 for each sex; p = 0.79).In both male and female HUVECs, treatment with Ang II significantly reduced migration to a similarl degree (by about 35% and 40% in MHU-VECs and FHUVECs, respectively) (Figure 2).

Effect of Ang II on Macroautophagy and Mitophagy
Macroautophagy and mitophagy were evaluated through the analysis of gene and protein expression of the main molecules involved in these processes.Under basal conditions, no significant differences between MHUVECs and FHUVECs were observed in the gene expression of Beclin-1, LC3I, LAMP-1, p62, or Parkin, while the protein expression of Beclin-1 was significantly higher in males (2.4 fold increase) and that of Parkin was higher in females (2.9 fold increase); the other proteins did not diverge (Figure 3).

Effect of Ang II on Macroautophagy and Mitophagy
Macroautophagy and mitophagy were evaluated through the analysis of gene and protein expression of the main molecules involved in these processes.Under basal conditions, no significant differences between MHUVECs and FHUVECs were observed in the gene expression of Beclin-1, LC3I, LAMP-1, p62, or Parkin, while the protein expression of Beclin-1 was significantly higher in males (2.4 fold increase) and that of Parkin was higher in females (2.9 fold increase); the other proteins did not diverge (Figure 3).Ang II treatment did not affect gene expression in MHUVECs (Figure 4), while it resulted in significant increases in Beclin-1 and Parkin levels in FHUVECs compared to baseline.Moreover, FHUVECs showed significantly higher levels of Parkin gene than MHUVECs (1.5 fold increase) (Figure 4).Ang II treatment did not affect gene expression in MHUVECs (Figure 4), while it resulted in significant increases in Beclin-1 and Parkin levels in FHUVECs compared to baseline.Moreover, FHUVECs showed significantly higher levels of Parkin gene than MHUVECs (1.5 fold increase) (Figure 4).In addition, in male cells, the treatment with Ang II caused an increase in p62 and LAMP-1 protein expression and a decrease in Parkin levels compared to basal conditions (Figure 5).A significant increase in the expression of Beclin-1 protein was instead found in Ang II-treated FHUVECs than in untreated cells (Figure 5).
Finally, the LC3 II/I ratio and LAMP-1 protein were significantly higher in MHUVECs than in FHUVECs (1.3 and 1.98 fold increase, respectively), while the Parkin protein was significantly more expressed in Ang II-treated FHUVECs than in male cells (2.6 fold increase) (Figure 5).
In addition, in male cells, the treatment with Ang II caused an increase in p62 and LAMP-1 protein expression and a decrease in Parkin levels compared to basal conditions (Figure 5).A significant increase in the expression of Beclin-1 protein was instead found in Ang II-treated FHUVECs than in untreated cells (Figure 5).Finally, the LC3 II/I ratio and LAMP-1 protein were significantly higher in MHU-VECs than in FHUVECs (1.3 and 1.98 fold increase, respectively), while the Parkin protein was significantly more expressed in Ang II-treated FHUVECs than in male cells (2.6 fold increase) (Figure 5).
Ang II affected the single miRNA level in a sex-dependent way (Figure 6): miR-126-3p was downregulated only in MHUVECs, while miR-133a-3p was upregulated only in FHUVECs.MiR-223-3p was downregulated by Ang II both in male and female cells, being the reduction more pronounced in FHUVECs.Finally, miR-29b-3p and miR-133b were not
Ang II affected the single miRNA level in a sex-dependent way (Figure 6): miR-126-3p was downregulated only in MHUVECs, while miR-133a-3p was upregulated only in FHUVECs.MiR-223-3p was downregulated by Ang II both in male and female cells, being the reduction more pronounced in FHUVECs.Finally, miR-29b-3p and miR-133b were not affected by Ang II.
Notably, sex influences both activities of endothelial cells and Ang II [20][21][22]25,46].The results of the present study indicate that some sex differences are present in basal cells, but the major novelties are linked to exposure to Ang II.In fact, the polypeptide induced qualitative and quantitative sex differences.In particular, for the first time, it has been demonstrated that Ang II induces cell autophagy in HUVECs in a sex-dependent manner.
In basal HUVECs, the only sex differences measured regard Parkin gene and protein, which are more expressed in FHUVECs than MHUVECs, and the expression of Beclin-1
Notably, sex influences both activities of endothelial cells and Ang II [20][21][22]25,46].The results of the present study indicate that some sex differences are present in basal cells, but the major novelties are linked to exposure to Ang II.In fact, the polypeptide induced qualitative and quantitative sex differences.In particular, for the first time, it has been demonstrated that Ang II induces cell autophagy in HUVECs in a sex-dependent manner.
In basal HUVECs, the only sex differences measured regard Parkin gene and protein, which are more expressed in FHUVECs than MHUVECs, and the expression of Beclin-1 protein, which is more elevated in MHUVECs than in FHUVECs.The higher expression of Beclin-1 in basal male cells confirms the previous data [25]; however, previously, it was found that the LC3II/LC3I ratio was higher in MHUVECs than in FHUVECs [25].Instead, here, the LC3II/LC3I ratio is similar in both FHUVECs and MHUVECs.The contradictory results could depend on the demographic characteristics of mothers.Here, they are older and weigh less than in the previous paper, and it is known that autophagic activity fits with metabolic profile and age [49,50].
Ang II elevates sex differences from two to five when compared to basal cells.Among the parameters that become different with sex, there is cell viability: in FHUVECs, there is a small but not significant decrease in cell viability, whereas in male cells, it is significantly increased.This is in line with previous HUVEC cell lines of unknown sex [51,52], but in the majority of cases, it was shown that Ang II induced viability loss in HUVEC cell lines of unknown sex (an exhaustive list of references about this point is reported in Table S3 [ [53][54][55][56][57][58][59][60][61][62][63][64][65][66]).The discrepancy could be attributed to the use of HUVEC cell lines versus the primary cultures we used, and to different methods of culture and viability determination.
Interestingly, both in basal and Ang II-stimulated FHUVECs there is an enhancement of autophagy occurring in a major elimination of the dysfunctional mitochondria.Several sex differences in female and male mitochondria are reported during postnatal life [67][68][69]; here, it is evident that mitochondrial sex differences start in prenatal life either in basal or Ang II-treated cells.In particular, Ang II promotes the expression of both the Beclin-1 and Parkin genes and proteins in FHUVECs, whereas the p62 and LAMP-1 proteins are similar in basal and Ang II-treated female cells.These findings suggest that Ang II in FHUVECs is a regulator of autophagy being the effect focused on the initiation of the process and on mitochondrial autophagy.This is in line with the fact that Ang II is a facilitator of the translocation of PARK2 into mitochondria, where it co-localizes into Parkin-positive mitochondria [70], suggesting a potential role for Beclin-1 in linking mitochondria to autophagosomes [71].Similar results were obtained in rat hearts [72] and in immortalized mouse podocytes of unknown sex [73].
In MHUVECs, Ang II increases the expression of LAMP-1 and p62 protein and decreases the expression of Parkin protein.The multifactorial protein p62 participates in the autophagic process as a lysophagy adapter [74], essential to the selective autophagy of protein aggregates and Parkin-independent mitochondria autophagy [75].In addition, the increase in p62 levels inside cells is associated with a defect in the final steps of autophagy [28].p62 binds to LC3 and co-localizes with LC3 and LAMP-1, so therefore may also be used to monitor the autophagic flux [28].The LAMP-1 protein is required for autophagosomes' fusion with endosomes/lysosomes [28].Globally, the influence of "male sex" is more evident when the final steps of the process are considered.
Ang II affects the levels of some miRNAs such as miR-126-3p (one of the most abundant miRNAs in endothelial cells involved in vascular remodeling [76,77]), whose levels are reduced only in MHUVECs, while miR-133a-3p (a cardiomyocytes-derived miRNAs and one of miRNA linked to angiogenesis [78]) is upregulated only in FHUVECs.Plasma miR-126 increases also in hypertensive patients with albuminuria, and miR-126-3p appears to be a good predictor of endothelial dysfunction, which is also associated with a shorter time free of both cardiovascular events, including coronary artery disease and stroke [79].
Previously, it was shown that expression of miR-133a-3p was reduced in a dosedependent manner by Ang II in a cell line of HUVECs of unknown sex inducing apoptosis [80].In line with previous data [81], miR-223-3p is expressed in freshly isolated endothelial cells.It is involved in invasiveness, immunity, and metabolism-related disorders such as diabetes mellitus [82,83].It is also considered an independent, non-invasive biomarker of atherosclerosis and/or type 2 diabetic patients [82].Its levels are reduced by Ang II both in male and female cells, indicating that it is a target of Ang II.Finally, miR-29b-3p (anti-fibrotic miRNA deriving from cardiac and fibroblast cells, and a key regulator of angiogenesis [84,85]) and miR-133b (a cardiomyocyte-derived miRNA and a factor involved in atherosclerosis [86]) were unchanged in both MHUVECs and FHUVECs.
The individual miRNAs may affect different steps of the autophagic process.The increase in levels of miR-133a-3p observed in FHUVECs is associated with an increase in the Parkin and Beclin-1 proteins, whereas the decrease in miR-126-3p in MHUVECs is associated with an increase in p62 and LAMP-1.Also, Yang et al. [33] described that some miRNAs (miR-376, miR-17, and miR-216a, for example) can inhibit the initial formation stage of autophagosomes, while others (miR-204, for example) inhibit the elongation stage.
In light of what has been reported, we can state that the relationship between miRNAs and organelle-specific autophagy after treatment with Ang II is influenced by sex.Thus, the effect of Ang II depends on a single miRNA and the sex of cells, indicating the strategic relevance of sex.A deeper knowledge of the influence of sex on the interaction between autophagy, miRNAs, and RAAS will give useful insights into the mechanisms of endothelial dysfunction and underline that further investigations will provide insights into innovative pharmacological approaches and biomarkers to target RAAS-mediated CVD and obtain a personalized treatment.

Donors
Umbilical cords were obtained from healthy Caucasian human male and female neonates vaginally delivered at term (37-42 weeks) at the Obstetrics and Gynaecology Clinic, University of Sassari.The mothers were healthy, non-obese, non-smoking, and drugfree, except for folic acid and iron supplementation, and had uncomplicated pregnancy.HUVECs were obtained only from umbilical cords of normal-weight neonates according to Bertino et al. [87] (2430-4050 and 2550-4190 g for males and females, respectively, which represented the 10th and 90th centiles in Ines charts), and they were isolated within 24 h of spontaneous delivery.Informed consent was obtained from the mothers of all subjects donating umbilical cords following the Declaration of Helsinki.

Experimental Model
The perturbation induced by Ang II in HUVECs is considered a good model for studying endothelial functions [42].Male and female basal groups (HUVECs cultivated without treatments) and male and female Ang II-treated HUVECs at concentrations indicated in tables and figures for 24 h were used.Some pilot experiments were performed to highlight whether Ang II had the same cytotoxic effects in male and female cells measuring lactate dehydrogenase (LDH) and lipid peroxidation as malondialdehyde (MDA) levels.
In addition, it was investigated whether the housekeeping gene glyceraldehyde-3phosphate dehydrogenase (GAPDH) has similar values in male and female cells.The values measured value were 18.27 ± 0.20 mean Ct for female cells, and 18.07 ± 0.30 for males (n = 5 for each sex; p = 0.21).Furthermore, pilot experiments showed that the housekeeping gene U6, which is normally used to normalize miRNAs data, did not differ between male and female cells (14.56 ± 0.84 mean Ct for female cells, and 14.68 ± 0.40 for male cells, n = 5 for each sex; p = 0.76).

Cell Isolation and Characterization
Umbilical cords were collected and stored at 4 • C and processed within 24 of collection.Primary female HUVECs (FHUVECs) and male HUVEC (MHUVECs) were isolated by collagenase treatment (Sigma-Aldrich, Milano, Italy), as previously described by Addis et al. [25] and cultured in plates pre-coated with 1% gelatine (Sigma-Aldrich, Milano, Italy) in an M199 medium (Life Technologies, Monza, Italy) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Monza, Italy), 10% newborn calf serum (NBCS) (Life Technologies, Monza, Italy), 1% antibiotic/antimycotic (Sigma-Aldrich, Milano, Italy) and 2 mM of L-glutamine (Sigma-Aldrich, Milano, Italy), until confluence, in a 5% CO 2 humidified atmosphere.As previously described (Addis), cultured cells were characterized as endothelial cells by the exhibition of cobblestone morphology when they were contactinhibited and by an evaluation of the expression of von Willebrand factor, a glycoprotein that is constitutively stored in intra-endothelial Weibel-Palade granules.
FHUVECs and MHUVECs were used in passage 3 to ensure their endothelial characteristics, and all experiments were conducted in duplicate or triplicate.

Cell Viability
FHUVECs and MHUVECs were seeded in a 96-well plate (about 15,000 cells/well in triplicate) and treated with Ang II (0.1-0.5-1 µM) for 24 h.Cell viability was determined by the crystal violet assay according to [88].The absorbance was recorded at 540 nm and the percentage of viability was calculated in comparison with basal cells, for which a value of vitality equal to 100% has been assumed.

LDH Assay
LDL release was measured in culture medium from basal and cells treated with Ang II (0.1-0.5-1 µM), using the LDH Cytotoxicity Detection kit (Roche Diagnostics, Monza, Italy), following the manufacturer's instructions.LDL release was expressed as the percentage of the LDH measured in the medium divided by the LDH release measured after cell treatment with 2% Triton X-100 (positive control, 100% LDH release).

Migration Assay
Migration was evaluated using the chemotaxis assay.HUVECs (6.0 × 10 4 cells/well) were suspended in M199 medium with or without Ang II (0.5 µM), and placed into the upper chamber of a 24-well modified Boyden Chamber (8.0 µm cell culture inserts; BD Bioscience, Milan, Italy).
Cells inserts with porous membranes were placed over a bottom chamber containing an M199 medium.After 24 h of incubation at 37 • C, the cells that had migrated to the lower side of the filter were stained with DAPI (0.2 mg/mL; Sigma-Aldrich, Milan, Italy), and 5 unit fields per filter were counted using a fluorescence microscope (Motic AE31, Kowloon City, Hong Kong).The percentage of migrated cells was calculated using the ImageJ software (Version 1.53t; NIH, Los Angeles, CA, USA), counting nuclei at each condition in comparison with the baseline.Each sample was examined in duplicate.

MDA Determination
MDA was determined in cell supernatants (100 µL) using the TBARs method as previously described [89] after 24 h of incubation with Ang II (0.5 µM).The quantification was performed spectrophotometrically at 535 nm by measuring the absorbance produced by 100 µL of the sample.Calibration curves were built with standards of MDA at 5, 10, 25, and 50 µM.
The relative mRNA expression levels were calculated by the 2 −∆Ct method and GAPDH mRNA levels were used for normalization.Gene-specific primer pairs are listed in Table S1.4.10.RNA Isolation, miRNAs Reverse Transcription, and Quantitative (q)PCR Analysis RNA from FHUVECs and MHUVECs were purified using the TriPure isolation reagent (Roche, Merk Life Science, Milano, Italy) following the manufacturer's guidelines.miRNA was reverse-transcribed using Mir-X miRNA FirstStrand Synthesis (Takara Bio San Jose, CA, USA) and subsequently analyzed by quantitative (q)PCR analysis using SYBR Green mix (Kapa Biosystems, Merk Life Science, Milano, Italy).Relative levels of miRNAs were calculated by the 2 −∆∆Ct method and U6 RNA levels were used for normalization.Genespecific primer pairs are listed in Table S2.

Statistical Analysis
Data were reported as the mean ± standard deviation (SD).A comparison between basal and Angiotensin II-treated cells and between male and female cells in each experimental condition was performed by a t-test using Sigma-Stat 3.1 software (Systat Software, Erkrath, Germany).
The distribution of samples was assessed by Kolmogorov-Smirnov and Shapiro tests.A p < 0.05 was considered statistically significant.

15 Figure 1 .
Figure 1.Linear regression analysis of the effect of angiotensin II on the viability of MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.

Figure 1 .
Figure1.Linear regression analysis of the effect of angiotensin II on the viability of MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.

Figure 2 .
Figure 2. Effect of Ang II on the migration of MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.Representative photographs were taken at ×20 magnification.

Figure 2 .
Figure 2. Effect of Ang II on the migration of MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.Representative photographs were taken at ×20 magnification.

.Figure 3 .
Figure 3. Densitometric analysis and representative Western blo protein expression in basal conditions in MHUVECs (light blue) reported as mean ± SD of at least 5 subjects per sex.

Figure 3 .
Figure 3. Densitometric analysis and representative Western blotting images of Beclin-1 and Parkin protein expression in basal conditions in MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.

Figure 3 .
Figure 3. Densitometric analysis and representative Western blotting images of Beclin-1 and Parkin protein expression in basal conditions in MHUVECs (light blue) and FHUVECs (orange).Data are reported as mean ± SD of at least 5 subjects per sex.

Figure 5 .
Figure 5. Densitometric analysis and representative Western blo ing images of LAMP-1, p62, Beclin-1, and Parkin proteins, and LC3 II/I ratio in MHUVECs (light blue) and FHUVECs (orange) before and after Ang II treatment.Data are reported as mean ± SD, calculated as an increase over the control of at least 5 subjects per sex.* p = 0.03 versus Ang II-treated MHUVECs.

Figure 5 .
Figure 5. Densitometric analysis and representative Western blotting images of LAMP-1, p62, Beclin-1, and Parkin proteins, and LC3 II/I ratio in MHUVECs (light blue) and FHUVECs (orange) before and after Ang II treatment.Data are reported as mean ± SD, calculated as an increase over the control of at least 5 subjects per sex.* p = 0.03 versus Ang II-treated MHUVECs.

Author
Contributions: F.F.: Conception and design of the study; data interpretation, drafting, and revision of the manuscript.G.C.: collected information about mothers and newborns, drafting and revision of the manuscript; G.D.: performed experiments; V.L. and M.L.I.: gene expression and miRNAs analysis; A.D.D.: collected umbilical cords; A.M.: drafting and revision of the manuscript; I.C.: Conception and design of study, analysis, and interpretation of data, drafting, and revision of the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This research was partially financed by the University of Sassari through the grants "FAR 2020" and "PRIN2017".Institutional Review Board Statement:This study was approved by the Independent Ethical Committee of Azienda Ospedaliero Universitaria (AOU Cagliari; prot.PG/2018/18480 of 20 December 2018).Informed Consent Statement: Each participant provided informed consent for the study.

Table 1 .
Baseline characteristics of mothers and newborns.