Studies have revealed that the language network of Broca’s area and Wernicke’s area is modulated by factors such as disease, gender, aging, and handedness. However, how occupational factors modulate the language network remains unclear.In this study, taking professional seafarers as an example, we explored the resting-state functional connectivity (RSFC) of the language network with seeds .The results showed seafarers had weakened RSFC of Broca’s area with the left superior/middle frontal gyrus and left precentral gyrus, and enhanced RSFC of Wernicke’s area with the cingulate and precuneus. Further, seafarers had a less right-lateralized RSFC with Broca’s area in the left inferior frontal gyrus, while the controls showed a left-lateralized RSFC pattern in Broca’s area and a right-lateralized one in Wernicke’s area. Moreover, seafarers displayed stronger RSFC with the left seeds of Broca’s area and Wernicke’s area.These findings suggest that years of working experience significantly modulates the RSFC of language networks and their lateralization, providing rich insights into language networks and occupational neuroplasticity. The human brain controls language , movemenResting-state functional magnetic resonance imaging (rsfMRI) is widely used to map the physiology and behavior of the healthy/diseased brain . FurtherIn this paper, taking seafarers as an example, two important sub-functions of language, namely, the language network (study 1) and the lateralization of the language network (study 2), were investigated to explore the association between the occupational factor and language by using the method of resting state functional connectivity (RSFC) . The anaSince seafarers have been engaged in repetitive technical work for a long time we recruited twenty male professional seafarers  from a shipping company in Shanghai, China. All of them had more than 10 years of experience in navigation. For non-seafarers, 20 Chinese male participants , were recruited from land-based jobs  at university or secondary school campuses. All the subjects in the non-seafarer group had no maritime professional training, maritime navigational skills, or long-term experience on the sea. All subjects signed the informed consent form and were considered to have normal functions of language and communication. Also, no history of mental health conditions or neurological diseases were reported. The blood-oxygen-level-dependent imaging (BOLD) rsfMRI data for each participant was scanned at the Shanghai Key Laboratory of Magnetic Resonance. All participants were informed about the purpose of the study and signed a written consent form according to the procedures approved by the IRB of East China Normal University (ECNU). The specific parameters were listed as follows: GE 3.0 Tesla using a gradient echo EPI, a total of 36 slices covering the whole brain area, 160 time points, TR (time of repetition) = 2 s, matrix size = 64 × 64, in-plane resolution = 3.75 mm × 3.75 mm, and slice thickness = 4 mm. The detailed information related to the dataset can also be found in 1 and the Resting-State fMRI Data Analysis Toolkit (REST).2 The preprocessing steps for the Resting-State fMRI data of each subject were as follows: (1) slice timing; (2) realignment; (3) normalization by EPI template (resampling voxel size = 3 mm*3 mm*3 mm); (4) spatial smoothing using a Gaussian kernel with FWHM = 6 mm; (5) nuisance regression including covariates such as six head motion parameters, whole brain mean signal, white matter signal, and cerebrospinal fluid signal; (6) band-pass temporal filtering (0.01–0.1 Hz); and (7) scrubbing volumes with sudden head motion, i.e., a threshold of frame-wise displacement (FD) was set to 0.05, and we removed one volume before and two volumes after the motion spike.All data preprocessing was performed using the Data Processing Assistant for RS-fMRI software package (DPARSF)  which isThe RSFC method generates a high-precision functional connection diagram of a complex brain system by interpreting the relevant patterns of low-frequency fluctuations in the blood oxygen level signals, which can be used to identify language-related functional tissues. In this paper, the Broca and Wernicke in the left side of brain were selected as the region of interest (ROI) (lBro and lWer), with MNI coordinates  and  as the center of the seed points  with a rt-test results  is defined by following formula (2)  :zFCL is a whole brain functional connection diagram based on the left seed points , respectively, and zFCflipped R is a left-right flipped functional connection diagram based on the right seed points (rBro and rWer), respectively. Similarly, as in previous studies  and the rBro and rWer and the right hemisphere (RH). Also, contralateral asymmetry was established on the right side of the AI map, indicating the differences between the lBro or lWer and the RH and the rBro and rWer and the LH. Further, a one-sample t-test was conducted to reveal regions which show significant hemispheric asymmetry based on individual AI maps. Moreover, the two-sample t-test was applied to analyze the differences of language lateralization between seafarers and the control participants. All RSFC maps and AI maps were established with the test criteria of p 0.005 and cluster size >200 voxels (corresponding to corrected pFWE 0.05).where  studies , ipsilatt-test analysis of the linguistic functional connectivity patterns from the seafarer and non-seafarer groups revealed that: the negative functional connectivity of Broca’s region appeared weaker in the seafarer group than the non-seafarer group, especially in the left superior/middle frontal gyrus and left precentral gyrus .A profile of the lateralization for each subject was obtained based on the RSFC map in terms of Broca’s area and Wernicke’s area. REST software was used to conduct a one-sample According to t-test analysis on the AI maps of the two core language regions for the two groups. The statistical results are shown in AI maps corresponding to the Broca’s region, the seafarer group elicited greater functional asymmetry in the paracentral and precuneus (BA7 and BA31). For the AI maps corresponding to the Wernicke’s area, the lateralization difference between the seafarer group and the non-seafarer group was mainly reflected in the left frontal gyrus and bilateral precuneus.In order to further quantify the differences in language lateralization between the seafarer group and the non-seafarer group, we performed a two-sample Previous studies have investigated whole-brain language networks using the RSFC method . SulpiziIn this study, we selected the special occupation group of seafarers as the research object, and compared the functional language network seafarers with non-seafarers. Regarding the seafarers, the functional connectivity related to functional language networks showed a negative connection with Broca’s area, though strongly left-lateralized, including in the left SFG/MFG (BA 6) and the precentral gyrus (BA 6 and 4), which may be involved due to the extended length of time spent in a relatively closed environment and the lack of spoken interaction. AI asymmetry in seafarers and non-seafarers. We found slightly rightward lateralization in the Broca’s seed of seafarers with left IFG, and almost leftward asymmetric distribution in the non-seafarers . The patients/participants provided their written informed consent to participate in this study.HW and DP: conceptualization, methodology, validation, formal analysis, and writing—original draft. HY: conceptualization, methodology, validation, formal analysis, writing—original draft, and funding acquisition. YY: investigation and writing—review and editing. WZ: investigation, writing—review and editing, and data curation. CC and NW: conceptualization, resources, writing—review and editing, supervision, funding acquisition, and project administration. All authors contributed to the article and approved the submitted version.


Renal cell carcinoma (RCC) is the most common type of kidney cancer. The therapeutic strategies are based on surgery and/or specific therapies able to inhibit growth factors that have been shown to promote the growth and spread of tumors. Currently, there is no established biomarker which helps in early diagnosis and in better disease monitoring with a high sensitivity. Much information could be provided by body fluids, especially blood liquid biopsy (LB), that are increasingly interesting to researchers. LB is a non- or minimally invasive procedure that could allow clinicians to monitor cancer evolution, also thanks to the presence of small vesicles known as extracellular vesicles (EVs) secreted by tumor cells and containing useful information. In particular, growing interest is focused on small RNA molecules (miRNAs) that are involved in tumor growth and could represent potential diagnostic, prognostic, and predictive biomarkers in RCC, as we summarize in this review.Renal cell carcinoma (RCC) is the second most common cancer of the urinary system. The current therapeutic strategies are based on partial or total nephrectomy and/or targeted therapies based on immune checkpoint inhibitors to which patients are often refractory. Preventive and screening strategies do not exist and the few available biomarkers for RCC are characterized by a lack of sensitivity, outlining the need for novel noninvasive and sensitive biomarkers for early diagnosis and better disease monitoring. Blood liquid biopsy (LB) is a non- or minimally invasive procedure for a more representative view of tumor heterogeneity than a tissue biopsy, potentially allowing the real-time monitoring of cancer evolution. Growing interest is focused on the extracellular vesicles (EVs) secreted by either healthy or tumoral cells and recovered in a variety of biological matrices, blood included. EVs are involved in cell-to-cell crosstalk transferring their mRNAs, microRNAs (miRNAs), and protein content. In particular, transferred miRNAs may regulate tumorigenesis and proliferation also impacting resistance to apoptosis, thus representing potential useful biomarkers. Here, we present the latest efforts in the identification of circulating miRNAs in blood samples, focusing on the potential use of EV-derived miRNAs as RCC diagnostic and prognostic markers. Renal cell carcinoma (RCC) is the second most lethal malignancy in the urological system  and reprThe incidence of the disease varies widely throughout the world and appears to be highest in more developed countries. White people have a lower risk of RCC than Native, African, and Hispanic Americans, probably due to the disparities in diets, lifestyles, socioeconomic conditions, education, and economical possibility for healthcare. Risk factors connected to RCC could be distinguished as (a) unmodifiable, such as age, race, and gender, and (b) modifiable, that include diet, alcohol, occupational exposure, and drugs . The peaUnmodifiable and modifiable risk factors are often related: RCC is doubly diagnosed in men where some modifiable risks such as hypertension, tobacco smoking, and obesity are more frequently observed. RCC originates from the kidney cortex, in particular from the epithelial cells of renal tubules ,4, and iRCC is a chemo- and radio-resistant neoplasia. The current therapeutic strategies are based on the surgical approach of radical and/or partial nephrectomy that remains the only effective therapy for clinically localized RCC . TraditiThose approaches target the peculiar hypervascularity of RCC and trigger immune cell infiltration by immune checkpoint inhibitors (ICIs), with or without antiangiogenic tyrosine kinase inhibitor (TKI)-based combinations . Thus, tICIs can stimulate the host immune response against cancer through PD1 inhibitors (Nivolumab and Pembrolizumab) or anticytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors (Ipilimumab). The first group can block the interaction between programmed cell death protein 1 (PD1) and programmed death-ligand 1 (PD-L1) able to downregulate the host T cells and, therefore, the immune response . Many stHowever, the response and tolerance to these combination treatments are not always satisfying, probably due to the lack of recognized molecular targets, an intratumoral heterogeneity, and different RCC molecular subtypes. The therapeutic strategy is based on the clinical prognostic score  and Memorial Sloan Kettering Cancer Center (MSKK) criteria), drug toxicities, patients’ comorbidities, and preference. In fact, nowadays the International mRCC Database Consortium risk model remains the only prospectively known predictive biomarker in mRCC .Although targeted therapies represent the standard treatment options for RCC, nearly all patients treated with targeted drugs will eventually experience disease progression. Unfortunately, a significant number of RCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilization nor clinical benefits, probably due to the intratumoral heterogeneity and molecular subtypes ,16.Therefore, the identification of new promising biomarkers in RCC is an urgent clinical need that leads clinicians and researchers to investigate the role of cell components as microRNAs (miRNAs) secreted by tumor cells and packaged into extracellular vesicles (EVs) released in biological fluids .For decades, surgical tissue biopsy has firmly been considered the gold standard for solid tumor diagnosis; however, it may become a diagnostic obstacle for its invasiveness, unrepeatability, and especially its peculiarity to immortalize that moment without taking into account the tumor heterogeneity and dynamism . The molOver the past decade, liquid biopsy (LB) has revolutionized the field of clinical oncology, providing ease in tumor sampling and continuous tumor monitoring .Thus, LB is gaining relevance in the clinical scenario of cancer for early diagnosis and treatment stratification, as well as residual disease detection and recurrence monitoring . In particular, overall survival in mRCC patients remains low despite the development of novel targeted therapeutic approaches. LBs could provide an attractive and noninvasive method to determine the risk of recurrence or metastatic dissemination during follow-up and, thus, improve the clinical outcomes and quality of life of RCC patients . SeveralThe identification of biomarkers in LB could enable the continuous and real-time collection of RCC patients’ information for diagnosis, prognosis assessment, and treatment response monitoring .Many studies have attempted to use LB as a routine method for the clinical diagnosis of RCC and for the prediction of patients’ grades, stages, and survival to identify patients at high risk for metastasis and recurrence ,27,28,29EVs and miRNAs are emerging as a platform with potentially broader and complementary applications in LB . EVs are small vesicles enclosed in a lipid bilayer, continuously released by all viable cells, in physiological and pathological conditions . AccordiEVs can transport tumor-derived material, including lipids, a wide variety of RNA , DNA, proteins, enzymes, and metabolites. They play a key role in cell-to-cell communication ; by tranThese small messengers are involved in tumorigenesis and tumor metastasis due to their widespread distribution throughout the blood and lymphatic circulation. Several studies indicate that tumor-released EVs may orchestrate tumor-progression-stimulating proliferation, angiogenesis, chemoresistance, and immune escape ,38,39. IRecently, EVs showed increasing potential in the field of urological malignancies . An incrThe recent knowledge is related to the biological role of EVs shed by RCC tumor cells in RCC progression, such as angiogenesis, immune escape, and tumor growth .Horie K et al. analyzed the hypoxic conditions in RCC that stimulate the release of tumor-cell-derived EVs. Their in vitro results, based on migration and tube formation assays, suggested the possibility that carbonic anhydrase 9 (CA9) enriched in exosomes and released from hypoxic RCC may enhance angiogenesis in the microenvironment, thereby contributing to cancer progression . In addiMoreover, specific protumorigenic roles have been attributed to EVs released by CD105+ renal CSCs, due to their cargo enriched in several proangiogenic mRNAs and miRNAs . Those cA specific role of renal-CSC-derived EVs has been additionally described for the formation of the premetastatic niche, which consists of a complex network of information exchanges. These EVs may sustain an unfavorable outcome for the tumor by enhancing tumor vascularization and by contributing to the establishment of a premetastatic niche . More reMicroRNAs (miRNAs) are small noncoding RNA molecules of 18–22 nucleotides capable of post-transcriptionally regulating protein translation, due to increased mRNA degradation ,38. For Over the last few years, the aberrant expression of miRNAs in cancer development has been dissected, identifying some miRNAs highly expressed in cancer cells with an oncogenic role and miRNAs acting as tumor suppressors. The deregulation of both groups of miRNAs contributes to tumor development mechanisms: oncogenic miRNAs (onco-miRNAs) are commonly overexpressed in cancerous tissues, and tumor suppressor miRNAs are conversely downregulated in tumors . Their sIn 2017, for the first time, Tian and colleagues compared the expression levels of miRNAs in plasma and in plasma-derived exosomes, showing no differences in healthy donors. However, in lung cancer patients, the onco-miRNAs were more enriched in exosomes than free in circulation, assuming an increased involvement of EVs in cancer for miRNA-based cell-to-cell communication .Cancer cells can actively load miRNAs into EVs for supporting tumor development and spread due to their peculiarity of protecting their cargo from the environment. The EV double layer allows the content to be preserved, thus improving miRNA’s half-life and stability . FurtherIn RCC, many efforts have been made in the research of circulating miRNAs in biological fluids, such as serum, plasma, and urine. Because urine can be easily collected, urinary miRNAs are attractive as they may be promising biomarkers in diseases affecting the urinary tract. Several studies demonstrated their stability in urine, particularly when carried by EVs. However, urine samples and, consequently, urinary miRNA detection may be influenced by several physiological and pathological conditions which make their study very challenging ,55.Due to the ease of sampling, better specificity of detection, and higher stability, most studies focused on serum and plasma miRNAs as potential noninvasive diagnostic, prognostic, or predictive biomarkers in RCC patients .The expression profile of several miRNAs has revealed their valid potential as diagnostic biomarkers in human RCC, since they are differentially expressed in tumors when compared with their normal counterpart, but also between primary and metastatic tumors . They haAn increasing challenge in recent studies is to find potential biomarkers able to improve sensitivity to chemoradiotherapy, predicting patient’s response and resistance to targeted therapy. Circulating miRNAs might also be involved in acquiring resistance to treatment in RCC. Their promising role as predictors of response to therapy may acquire considerable importance in RCC, providing clinicians with crucial information to determine the optimal treatment plan and, thus, avoiding severe side effects of ineffective overtreatment  Figure .We reviewed the studies on free or encapsulated miRNAs differentially expressed in the blood samples of RCC patients compared to normal controls, as summarized in Tusong et al. reported the overexpression of miR-21 and miR-106a in RCC patients’ serum compared to healthy controls and their significant reduction a month after surgery compared with the preoperative group. This outcome suggests the potential diagnostic and predictive role of serum miR-21 and miR-106a as noninvasive biomarkers for RCC . MiR-21 Cheng et al. showed an upregulation of miR-21, miR-34a, and miR-224 and a downregulation of miR-141 in the sera of patients with ccRCC, reporting consistency in their expression profile with those of the corresponding tumor tissue samples .Interestingly, only the miR-21 expression levels in the serum of the patients were correlated with the patients’ clinical stage, while miR-224 expression levels were correlated with gender. However, this study showed that miR-34a, miR-224, miR-21, and miR-141 could be considered as potential ccRCC tumor markers .A diagnostic role has been attributed to several miRNAs, such as: miR-625-3p, miR-508-3p, and miR-885-5p, dysregulated in the serum of ccRCC patients ,62; miR-The late diagnosis of RCC, primarily due to a lack of early-stage diagnosis, encouraged researchers to discover novel miRNA signatures that discriminate RCC patients from healthy controls with a high degree of accuracy. In particular, in 2015, Whang et al. focused on a 5-miRNA panel including miR-28-5p, miR-362, miR-572, miR-193a-3p, and miR-378, demonstrating their potential value as an ancillary clinical diagnostic tool to also detect early-stage RCC, for which surgery is most effective . Next toMore recently, the upregulation of miR-765 was described in the plasma of ccRCC patients after tumor resection, suggesting its tumor suppressor role and identifying the proteolipid protein 2 (PLP2) as a candidate downstream target gene . In the In contrast to other studies, Heinemann et al. did not only compare the serum of patients with ccRCC and healthy donors, but also a group of patients with benign renal tumors (BRTs). As a result, the downregulation of miR-122-5p and miR-206 in the serum of both ccRCC and BRT groups compared to controls suggested their doubtful role in diagnosis. However, they demonstrated their prognostic value, showing that high levels of miR-122-5p and miR-206 were associated with a significantly shorter period of progression-free, cancer-specific, and overall survival . Plasma The comparison of plasma of ccRCC patients with localized and metastatic disease before and after surgery suggested that EV content varies depending on the presence or absence of the disease. An increased level of miR-301a-3p and a decreased expression of miR-1293 may be potential biomarkers of metastatic disease . Two stuRCC is considered one of the most unfavorable tumor diseases due to the late diagnosis and the poor prognosis. Nowadays, specific biomarkers validated for RCC early detection are not available and the actual treatments are often unable to avoid recurrence of the disease. LB could provide an attractive and noninvasive tool to assist the research for biomarkers in RCC tumors to capture a larger amount of the molecular heterogeneity compared to tissue biopsy and give prompt information on the risk of recurrence/relapse during follow-up.Many efforts are directed towards the research for circulating miRNAs that play a role in almost all aspects of cancer biology and development. In particular, miRNAs packaged in EVs and released in blood flow could allow expanding the spectrum of potential biomarkers for future use in the diagnosis and prognosis of RCC and in the prediction of therapeutic response.However, nowadays, there are no miRNAs widely applied as biomarkers in the clinical setting, partly due to the lack of isolation and quantification standardized protocols, the heterogeneity of study cohorts, and the variety of body fluids under investigation. Several studies have analyzed plasma- and serum-derived miRNAs, without focusing on the vesicle compartment. EV-derived miRNAs appear to be more stable than free miRNAs as EVs seem to protect them from degradation by macrophages. The EVs’ double-layered membrane and nanoscale size grant the miRNAs stability, thus prolonging their circulation half-life and enhancing their biological activity . After rNonetheless, beyond these technical limitations, the most exciting but challenging application will be to utilize EVs and their cargo as a clinical tool to diagnose and monitor disease. Thus, EV-derived miRNAs could provide a novel therapeutic tool in RCC clinical practice, where the lack of adequate biomarkers makes functional investigations urgently needed.


The mechanical environment of endothelial cells (ECs) in PAD is characterized by disturbed blood flow (d-flow) and stiff extracellular matrices. In PAD, the stiffness of arteries is due to decreased elastin function and increased collagen content. These flow and stiffness parameters are largely missing from current models of PAD. It has been previously proven that ECs exposed to d-flow or stiff substrates lead to proatherogenic pathways, but the effect of both, d-flow and stiffness, on EC phenotype has not been fully investigated. In this study, we sought to explore the effect of sex on proatherogenic pathways that could result from exposing endothelial cells to a d-flow and stiff environment. We utilized the scRNA-seq tool to analyze the gene expression of ECs exposed to the different mechanical conditions both in vitro and in vivo. We found that male ECs exposed to different mechanical stimuli presented higher expression of genes related to fibrosis and d-flow in vitro. We validated our findings in vivo by exposing murine carotid arteries to d-flow via partial carotid artery ligation. Since women have delayed onset of arterial stiffening and subsequent PAD, this work may provide a framework for some of the pathways in which biological sex interacts with sex-based differences in PAD.Peripheral arterial disease (PAD) is an age-related medical condition affecting mostly muscular arteries of the limb. It is the 3 PAD most commonly affects the older populations, with an incidence of 12%–20% in those older than 65 years . The onsecade 30–0 s, but to males , 8. Prevto males . The endto males . Our resh factor , 13.in vitro and in vivo studies have been done to understand the molecular pathways that lead to atherosclerosis  were less impacted than male HUVECs when exposed to higher and lower laminar shear stress and increasing substrate stiffness since they presented no change in morphology and YAP1 nuclear translocation (in vitro response of human aortic ECs (HAECs) in the context of PAD conditions (stiff substrate and d-flow). Given the powerful independent effect of stiffness or flow on ECs, we hypothesized that scRNA-seq would reveal sex-based phenotypic differences under PAD biomechanical conditions that may contribute to sex-based differences in PAD.Despite the NIH mandate to include sex as a biological variable, sex-differences have not been adequately investigated in this space , 18. Thelocation . In para2.2.1.2) while HAECs grown in the inner circle mimicked disturbed flow . The cells were sheared for 96 h utilizing an orbital shaker at 100 rpm, changing media every two days. For comparison, HAECs cultured on tissue culture plastic (TCP) immediately prior to subculture conditions above were analyzed. ECs on TCP (2 GPa) and static conditions are known to mimic many of the pathways seen under s-flow conditions, including THBS1  and two female (ages 46 and 53) donors  were expanded, cultured, and sheared in complete endothelial cell growth medium . HAECs of passages 4–6 were seeded onto modified 6-well plates functionalized with collagen-coated hydrogels  of different stiffnesses (25 kPa and 100 kPa) that mimic the EC environment in physiologic (25 kPa) and PAD conditions (100 kPa) , 21. The2.2.Following exposure to shear stress, HAECs were recovered utilizing TrypLE  and suspended in 1X PBS (Cat.# MT21040CV) with 0.04% BSA. After ensuring the proper cell concentration was collected, the samples were delivered to the Emory Integrated Genomics Core for single-cell processing and barcoding with the 10X Genomics Chromium device. NovaSeq S4 was utilized to prepare the libraries and sequence the samples. Then, the CellRanger software was utilized to demultiplex and align the sequenced data.2.3.scRNA-seq data was processed and analyzed using the Seurat package in R (RRID:SCR_016341). To remove low-quality cells or cell multiplets, standard quality control filters were applied . Cells c2.4.in situ control. D-flow has been shown to induce arterial stiffening similar to that seen in aged 80-week-old animal . The gene analysis for the in vivo validation was presented as the fold change normalized against 18S.Partial carotid artery ligation (PCL) surgery was performed on 9 to 15-week-old S129 and C57BL/6 wild type (WT) mice as previously published . The lefd animal . 2–3 mou2.5.p-value of <0.05.The validation data collected from quantitative PCR was analyzed utilizing GraphPad Prism. Each data point represents 2–3 LCAs or RCAs from mouse following 24 h of PCL. The fold change gene expression of the two WT strains utilized here (S129 and C57BL/6) were pooled for plotting. A two-sample Wilcoxon-Mann–Whitney test was done to compare between biological sex. Significance was set as a 3.3.1.XIST and RPS4Y1 rank among the highest in female and male, respectively. However, other non-sex-linked genes were identified. Specifically, in male HAECs, genes like VWF and BMP4 are differentially expressed, while in female HAECs, PIEZO2 was increased.To identify sex-specific differences in static conditions, HAECs on TCP and under static culture were pooled across donors. This condition mimics how ECs are typically expanded. After pooling all donors, eight distinct clusters were identified following unsupervised clustering and are shown using a UMAP plot . The clu3.2.CCN1, CCN2, COL8A1 and d-flow associated genes like THBS1 that present higher expression in male HAECs after mechanical stimuli.Motivated by the sex-specific differences shown in the static culture group, we next investigated the existence of sex-specific differences when HAECs were exposed to different magnitudes of shear stress and substrate stiffness. The relative gene expression across all shear stress and substrate conditions per donor was plotted in violin plots . Of inte3.3.in vitro scRNA-seq findings with two commonly used WT murine strains (S129 and C57BL/6). Here, EC-enriched RNA was collected after 24 h of d-flow, induced by PCL in the LCA; the contralateral RCA serving as an in situ control . After finding distinct differences between male and female HAECs on TCP under static culture conditions, we considered the effect of mechanical stimuli on sex-specific differences. We found that male HAECs presented higher expression of genes related to fibrosis and d-flow. Lastly, we validated our scRNA-seq results with CCN2, TAGLN, and THBS1 , which is similar in donor number to many publications –28. All TAGLN, TAGLN2, MYL9, and TPM2 in male and female HAECs  to HUVECs seeded on substrates of different stiffnesses (10 kPa or 100 kPa), they discovered that female HUVECs remained mostly invariant to the different combinations of mechanical stimuli suggesting that females might be more resistant to changes in the mechanical environment of ECs (TGF-β) has been proposed to be important to atherosclerotic remodeling, but the data presented was all from male animals (THBS1 can activate TGF-β, this mechanism may be relatively more important in males compared to females. Biological sex-based differences are present in cardiovascular diseases such as pulmonary arterial hypertension, aortic valve stenosis, PAD, and more (Biological sex is underreported in ar field , 18. In t of ECs . Here, w animals . Since Tand more , 26, 36.and more , 38. Forand more . The effin vitro model utilized in this work also enables rigorous delineation of sex-based EC differences in a setting that can exclude hormonal interactions of in vivo studies as well as comparisons between humans and mice that have distinct hormonal patterns across all ages. Still, this in vitro platform could be used to incorporate exogenous hormone levels to test in vivo conditions. Further, our in vivo validation supports acute sex-based EC expression differences under PAD flow conditions.This work highlights significant sex-based EC phenotypic differences induced by various clinically relevant culture conditions that may have profound impact on our understanding of sex-based clinical differences. In particular, this study found that male HAECs on TCP and cultured under static conditions and HAECs exposed to OS conditions had relatively similar gene expression in markers of EndMT and mesenchymal cells. In contrast, female HAECs showed higher EndMT and mesenchymal cell markers in HAECs cultured on static conditions when compared to other shear and substrate stiffness conditions. The In conclusion, sex-based differences are seen in HAECs under PAD conditions. These EC phenotypes may contribute to sex-based clinical differences in PAD. The use of scRNA-seq is a powerful technique that will allow better understanding of ECs under complex biomechanical conditions. Future work may further aid in the discovery of EC heterogeneity, genetic differences of PAD patients, as well as sex-based donor differences under static culture and under clinically relevant conditions.