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After birth, tissues grow continuously until reaching adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and (stem or non-stem) cell sources. Using a suite of experimental and computational multiscale approaches, we found that aortic expansion is guided by specific biological principles and scales with the vertebral column rather than animal body weight. Expansion proceeds via two distinct waves of arterial cell proliferation along blood flow that are spatially stochastic, yet temporally coordinated. Each wave exhibits unique cell cycle kinetics and properties, with the first wave exhibiting cell cycle durations as fast as 6 hours. Single-cell RNA sequencing showed changes in fatty acid metabolism concomitant with an increase in cell size. Mathematical modeling and experiments indicated endothelial cell extrusion is essential for homeostatic aortic growth and balancing excess proliferation. In a genetic model of achondroplasia, the aorta achieves proper scaling through enhanced cell extrusion while maintaining normal proliferation dynamics. Collectively, these results provide a blueprint of the principles that orchestrate aortic growth which depends entirely on differentiated cell proliferation rather than resident stem cells.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

MicroRNAs (miRNA) are major regulators of intercellular communication and key players in the pathophysiology of cardiovascular disease. This study aimed to determine the miRNA fingerprint in a cohort of 53 patients with acute myocardial infarction (AMI) with non-ST-segment elevation (NSTEMI) relative to miRNA expression in healthy controls (n = 51). miRNA expression was initially profiled by miRNA array in the serum of patients undergoing cardiac catheterization during NSTEMI (n = 8) and 1 year past the event (follow-up, n = 8) and validated in the entire cohort. In total, 58 miRNAs were differentially expressed during AMI (p < 0.05), while 36 were modified at follow-up (Fisher’s exact test: p = 0.0138). Enrichment analyses revealed differential regulation of biological processes by miRNA at each specific time point (AMI vs. follow-up). During AMI, the miRNA profile was associated mainly with processes involved in vascular development. However, 1 year after AMI, changes in miRNA expression were partially related to the regulation of cardiac tissue morphogenesis. Linear correlation analysis of miRNA with serum levels of cytokines and chemokines revealed that let-7g-5p, let-7e-5p, and miR-26a-5p expression was inversely associated with serum levels of pro-inflammatory cytokines TNF-α, and the chemokines MCP-3 and MDC. Transient transfection of human endothelial cells (HUVEC) with let-7e-5p inhibitor or mimic demonstrated a key role for this miRNA in endothelial function regulation in terms of cell adhesion and angiogenesis capacity. HUVEC transfected with let-7e-5p mimic showed a 20% increase in adhesion capacity, whereas transfection with let-7e-5p inhibitor increased the number of tube-like structures. This study pinpoints circulating miRNA expression fingerprint in NSTEMI patients, specific to the acute event and changes at 1-year follow-up. Additionally, given its involvement in modulating endothelial cell function and vascularization, altered let-7e-5p expression may constitute a therapeutic biomarker and target for ischemic heart disease.

Background/aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol., Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA-mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software., Results: miRNA-target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA-target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways., Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg., (©2020 American Association for Cancer Research.)

Despite the promising value of miRNAs in the diagnostic and prognostic of cardiovascular disease (CVD), recent meta-analyses did not support their potential. Methodological variances in studies may interfere with miRNA profile and affect their results. This study determines if the blood starting material is a source of variance in miRNA profile by performing a paired comparison in plasma and serum of the expression of primary miRNAs associated with CVD. Circulating miRNA yield was similar in both plasma and serum, although a significant increase was observed in patients with Non-ST-elevation myocardial infarction (NSTEMI) compared to control volunteers. When normalized by the expression of miR-484, different patterns of miRNA expression between serum and plasma. Although NSTEMI modified the expression of miR-1 and miR-208 in both serum and plasma, plasma displayed a higher variance than serum (Levene's test p < 0.01). For miR-133a and miR-26a, differences were only detected in serum (p = 0.0240), and conversely, miR-499a showed differences only in plasma of NSTEMI (p = 0.001). Interestingly, miR-21 showed an opposite pattern of expression, being increased in serum (2 -ΔΔCt : 5.7, p = 0.0221) and decreased in plasma (2 -ΔΔCt : 0.5, p = 0.0107). Plasma and serum exhibit different patterns of circulating miRNA expression in NSTEMI and suggest that results from studies with different starting material could not be comparable.

Women show a lower incidence of cardiovascular diseases than age-matched men, but this benefit disappears after menopause. Oestrogen-mediated vascular actions are mainly attributed to oestradiol and exerted by oestrogen receptors (ERα, ERβ and G protein-coupled oestrogen receptor), through rapid and/or genomic mechanisms, but these effects depend on ageing and inflammation. A cardiovascular approach in women's health has arisen due to controversy regarding oestrogen's beneficial impact as reported in experimental and observational studies and large randomized trials. These can be explained, in part, by two mutually non-exclusive hypotheses. On the one hand, the timing hypothesis, which states that oestrogen-mediated benefits occur before the detrimental effects of ageing are established in the vasculature; on the other hand, ageing and/or hormonal-associated changes in ER expression that could lead to a deleterious imbalance in favour of ERβ over ERα, generally associated with higher inflammation and endothelial dysfunction. In experimental studies, oestradiol acting on ERα promotes the release of vasoactive compounds such as nitric oxide (NO) and prostacyclin, and shifts the angiotensin axis towards angiotensin 1-7 production. Mechanisms underlying oestradiol vascular function also include anti-inflammatory and epigenetic modifications. 17β-Oestradiol changes the transcriptomic profile of endothelial cells, and the involvement of miRNA in the regulatory pathways of vascular function reinforces assumptions regarding the vascular actions of oestrogen. Thus, the present Symposium Review aims to postulate the role of ERα in oestrogen modulation of endothelium-derived mediators and vascular physiology, as well as its relationship with miRNA and inflammation, and elucidate how physiological changes in postmenopausal women counteract the observed effects., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Aims: Endothelial progenitor cells (EPC) play a role in endothelium integrity maintenance and regeneration. Decreased numbers of EPC or their impaired function correlates with an increase in cardiovascular events. Thus, EPC are important predictors of cardiovascular mortality and morbidity. Microparticles carrying Sonic hedgehog (Shh) morphogen (MPShh+) trigger pro-angiogenic responses, both in endothelial cells and in ischaemic rodent models. Here, we propose that MPShh+ regulates EPC function, thus enhancing vasculogenesis, and correcting the defects in dysfunctional EPC obtained from acute myocardial infarction (AMI) patients., Methods and Results: The mechanisms underlying Shh pathway function and nitric oxide (NO) production in EPC were evaluated. MPShh+ increased both the in vitro and in vivo vasculogenic capacity of EPC isolated from adult human peripheral blood samples. MPShh+ treatment significantly increased the expression of Shh signalling pathway genes (PTCH1, SMO, and GLI1) and masters of pro-angiogenic genes (NOS3, VEGFA, KDR, and KLF2) in EPC. Moreover, MPShh+ increased both the protein expression and activity of eNOS, resulting in increased NO production. Most importantly, MPShh+ improved the vasculogenic capacity of EPC from AMI patients to levels similar to that of EPC from healthy patients. All these effects were due to the activation of Shh pathway., Conclusion: MPShh+ increase both the vasculogenesis of EPC and their capacity to produce NO, including EPC from patients who have recently suffered an AMI. This study emphasizes MPShh+ and EPC as potential therapeutic tools for improving vascular regeneration as a treatment for cardiovascular ischaemic disease.

Cardiovascular diseases are a worldwide health problem and are the leading cause of mortality in developed countries. Together with experimental data, the lower incidence of cardiovascular diseases in women than in men of reproductive age points to the influence of sex hormones at the cardiovascular level and suggests that estrogens play a protective role against cardiovascular disease and that this role is also modified by ageing. Estrogens affect cardiovascular function via their specific estrogen receptors to trigger gene expression changes at the transcriptional level. In addition, emerging studies have proposed a role for microRNAs in the vascular effects mediated by estrogens. miRNAs regulate gene expression by repressing translational processes and have been estimated to be involved in the regulation of approximately 30% of all protein-coding genes in mammals. In this review, we highlight the current knowledge of the role of estrogen-sensitive miRNAs, and their influence in regulating vascular ageing.

The beneficial effects of estrogen on the cardiovascular system have been reported extensively. In fact, the incidence of cardiovascular diseases in women is lower than in age-matched men during their fertile stage of life, a benefit that disappears after menopause. These sex-related differences point to sexual hormones, mainly estrogen, as possible cardiovascular protective factors. The regulation of vascular function by estrogen is mainly related to the maintenance of normal endothelial function and is mediated by both direct and indirect gene transcription through the activity of specific estrogen receptors. Some of these mechanisms are known, but many remain to be elucidated. In recent years, microRNAs have been established as non-coding RNAs that regulate the expression of a high percentage of protein-coding genes in mammals and are related to the correct function of human physiology. Moreover, within the cardiovascular system, miRNAs have been related to physiological and pathological conditions. In this review, we address what is known about the role of estrogen-regulated miRNAs and their emerging involvement in vascular biology., Competing Interests: The authors declare no conflict of interest.

Background/aims: Estrogen signalling plays an important role in vascular biology as it modulates vasoactive and metabolic pathways in endothelial cells. Growing evidence has also established microRNA (miRNA) as key regulators of endothelial function. Nonetheless, the role of estrogen regulation on miRNA profile in endothelial cells is poorly understood. In this study, we aimed to determine how estrogen modulates miRNA profile in human endothelial cells and to explore the role of the different estrogen receptors (ERα, ERβ and GPER) in the regulation of miRNA expression by estrogen., Methods: We used miRNA microarrays to determine global miRNA expression in human umbilical vein endothelial cells (HUVEC) exposed to a physiological concentration of estradiol (E2; 1 nmol/L) for 24 hours. miRNA-gene interactions were computationally predicted using Ingenuity Pathway Analysis and changes in miRNA levels were validated by qRT-PCR. Role of ER in the E2-induced miRNA was additionally confirmed by using specific ER agonists and antagonists., Results: miRNA array revealed that expression of 114 miRNA were significantly modified after E2 exposition. Further biological pathway analysis revealed cell death and survival, lipid metabolism, reproductive system function, as the top functions regulated by E2. We validated changes in the most significantly increased (miR-30b-5p, miR-487a-5p, miR-4710, miR-501-3p) and decreased (miR-378h and miR-1244) miRNA and the role of ER in these E2-induced miRNA was determined. Results showed that both classical, ERα and ERβ, and membrane-bound ER, GPER, differentially regulated specific miRNA. In silico analysis of validated miRNA promoters identified specific ER binding sites., Conclusion: Our findings identify differentially expressed miRNA pathways linked to E2 in human endothelial cells through ER, and provide new insights by which estrogen can modulate endothelial function., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The Mas receptor is involved in the angiotensin (Ang)-(1-7) vasodilatory actions by increasing nitric oxide production (NO). We have previously demonstrated an increased production of Ang-(1-7) in human umbilical vein endothelial cells (HUVEC) exposed to estradiol (E2), suggesting a potential cross-talk between E2 and the Ang-(1-7)/Mas receptor axis. Here, we explored whether the vasoactive response and NO-related signalling exerted by E2 are influenced by Mas. HUVEC were exposed to 10nM E2 for 24h in the presence or absence of the selective Mas receptor antagonist A779, and the estrogen receptor (ER) antagonist ICI182780 (ICI). E2 increased Akt and endothelial nitric oxide synthase (eNOS) mRNA and protein expression, measured by RT-PCR and Western blot, respectively. Furthermore, E2 increased Akt activity (determined by the levels of phospho-Ser 473 ) and eNOS activity (by the enhanced phosphorylation of Ser 1177 , the activated form), resulting in increased NO production, which was measured by the fluorescence probe DAF-2-FM. These signalling events were dependent on ER and Mas receptor activation, since they were abolished in the presence of ICI or A779. In ex-vivo functional experiments performed with a small-vessel myograph in isolated mesenteric vessels from wild-type mice pre-contracted with noradrenaline, the relaxant response to physiological concentrations of E2 was blocked by ICI and A779, to the same extent to that obtained in the vessels isolated from Mas-deficient. In conclusion, E2 induces NO production and vasodilation through mechanisms that require Mas receptor activation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Intracellular renin-angiotensin system (RAS) can operate independently of the circulating RAS. Estrogens provide protective effects by modulating the RAS. Our aim was to investigate the effect of estradiol (E2) on angiotensin converting enzymes (ACE) 1 and ACE2 expression and activities in human endothelial cells (HUVEC), and the role of estrogen receptors (ER). The results confirmed the presence of active intracellular RAS in HUVEC. Physiological concentrations of E2 induced a concentration-dependent increase of ACE1 and ACE2 mRNA expression and ACE1, but not ACE2, protein levels. ACE1 and ACE2 enzymatic activities were also induced with E2. These effects were mediated through ERα activation, since ER antagonists ICI 182780 and MPP completely abolished the effect of E2. Moreover, the ERα agonist PPT mirrored the E2 effects on ACE1 and ACE2 protein expression and activity. Exposure of endothelial cells to E2 significantly increased Ang-(1-7) production. In conclusion, E2 increases Ang-(1-7) production, through ERα, involving increased ACE1 and ACE2 mRNA expression and activity and ACE1 protein levels., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The present study investigated the time-course for aging-associated effects on contractile and relaxing vascular responses and nitric oxide (NO) production in the aorta from female senescence-accelerated resistant (SAMR1) and prone (SAMP8) mice. Both SAMR1 and SAMP8 were studied at three different ages: 3 (young), 6 (middle age) and 10 (old) months. Concentration-response curves to phenylephrine (10(-8) to 10(-5) M) or acetylcholine (10(-9) to 10(-5) M) were performed in the aortic rings in the absence or in the presence of NO synthase (NOS) inhibitor L-NAME (10(-4) M). Protein and gene expression for endothelial NOS (eNOS) was determined by immunofluorescence, Western blot and real-time PCR. Although we have not seen any difference in vascular responses when comparing both strains at 3 months old, we found a significant aging-associated impairment of vascular reactivity that follows a distinct time-course in SAMR1 and SAMP8. In SAMR1, increases in phenylephrine contraction and decreases in acetylcholine relaxation were only seen at 10 months old, while SAMP8 displays altered responses at 6 months that are further impaired at 10 months old. L-NAME treatment enhanced phenylephrine contractions and completely inhibited acetylcholine relaxations in all age groups of SAMR1 and SAMP8. However, the magnitude of increase in phenylephrine contraction by L-NAME was markedly reduced by aging and followed a faster pace in SAMP8. Similar pattern of responses was observed in the time course for changes of eNOS expression, suggesting an earlier and more pronounced aging-associated decrease of NO production and eNOS expression in SAMP8. These results reveal that aging enhances contractile responses to phenylephrine and decreases endothelium-dependent relaxation to acetylcholine in the aorta from female mice by a mechanism that involves a decrease of NO production. This process occurs earlier in the aorta from SAMP8 mice, establishing these mice as suitable model to study cardiovascular aging in a convenient and standard time course., (© 2013.)