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Obstructive sleep apnea (OSA) is characterized by intermittent hypoxic environments at the cellular level and is an independent risk factor for the development of cardiovascular disease. Endothelial cell (EC) dysfunction precedes the development of cardiovascular disease; however, the mechanisms by which ECs respond to these intermittent hypoxic events are poorly understood. To better understand EC responses to hypoxia, we examined the effects of sustained hypoxia (SH) and intermittent hypoxia (IH) on the activation of HIF-1α in ECs. While SH stabilized HIF-1α and led to its nuclear localization, IH did not activate HIF-1α and the expression of its target genes. Using RNA-sequencing, we evaluated transcriptional responses of ECs to hypoxia. SH induced the expression of HIF-1α and hypoxia response genes, while IH affected cell-cycle regulation genes. A cytoscape protein-protein interaction network for EC response to hypoxia was created with differentially expressed genes. The network comprises cell-cycle regulation, inflammatory signaling via NF-κB and response to VEGF stimulus subnetworks on which SH and IH had distinct activities. As OSA is associated with elevated catecholamines, we investigated the effect of epinephrine on the EC response to SH and IH. Transcriptomic responses under IH and epinephrine revealed protein-protein interaction networks emphasizing distinct subnetworks, including cytokine-mediated TNFα signaling via NF-κB, Wnt/LRP/DKK signaling and cell cycle regulation. This study reveals differential transcriptomic responses under SH and IH characterised by HIF-1α transcriptional response induced only by SH, but not by IH. The study also features the potential molecular events that may occur at the vascular level in OSA.

The endothelialization of cardiovascular implants is supposed to improve the long-term patency of these implants. In addition, in previous studies, it has been shown, that the conditioning of endothelial cells by dynamic cultivation leads to the expression of an anti-thrombogenic phenotype. For the creation of a tissue-engineered vascular graft (TEVG), these two strategies were combined to achieve optimal hemocompatibility. In a clinical setup, this would require the transfer of the already endothelialized construct from the conditioning bioreactor to the patient. Therefore, the reversibility of the dynamic conditioning of the endothelial cells with arterial-like high shear stress (20 dyn/cm 2 ) was investigated to define the timeframe (tested in a range of up to 24 h) for the perseverance of dynamically induced phenotypical changes. Two types of endothelial cells were compared: endothelial colony-forming cells (ECFCs) and human aortic endothelial cells (HAECs). The results showed that ECFCs respond far more sensitively and rapidly to flow than HAECs. The resulting cell alignment and increased protein expression of KLF-2, Notch-4, Thrombomodulin, Tie2 and eNOS monomer was paralleled by increased eNOS and unaltered KLF-2 mRNA levels even under stopped-flow conditions. VCAM-1 mRNA and protein expression was downregulated under flow and did not recover under stopped flow. From these time kinetic results, we concluded, that the maximum time gap between the TEVG cultivated with autologous ECFCs in future reactor cultivations and the transfer to the potential TEVG recipient should be limited to ∼6 h., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sebastian Heene reports were provided by Leibniz University Hannover. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we investigated gene expression in vitro of human primary Aortic smooth muscle cells (AoSMCs) in response to 9% physiological dynamic stretch over a 4 to 72-h timeframe using RT-qPCR. AoSMC were derived from primary culture and were exposed to continuous cycles of stretch and relaxation at 1 Hz by a computer-controlled Flex Jr.™ Tension System. Unstretched control AoSMCs were simultaneously cultured in the same dishes. Our results revealed a rapid and significant upregulation of specific genes (COL1A1, FBN1, LAMA5, TGFBR1 and TGFBR2) within the initial 4 h for AoSMCs subjected to dynamic stretching, whilst control cells did not respond within the same 4 h. The upregulated genes were the ones associated with extracellular matrix (ECM) fibrillogenesis and regulation of traction forces. Interestingly, stretched cells maintained stable gene expression between 4 and 72 h, whilst control cells exhibited variations over time in the absence of mechanical cues. These findings shed light on the essential role played by pulsatile stretches in the regulation of gene expressions by AoSMCs and the intricate processes governing their mechanobiological function, paving the way for further investigations in cardiovascular health., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Calcium deposition in vascular smooth muscle cells (VSMCs), a form of ectopic ossification in blood vessels, can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that CCAAT/enhancer-binding protein beta (C/EBPβ) potentiates calcium deposition in VSMCs and mouse aorta induced by inorganic phosphate (Pi) or vitamin D 3 . Based on cDNA microarray and RNA sequencing data of Pi-treated rat VSMCs, C/EBPβ was found to be upregulated and thus selected for further evaluation. Quantitative RT-PCR and Western blot analysis confirmed that C/EBPβ was upregulated in Pi-treated A10 cells, a rat VSMC line, as well as vitamin D 3 -treated mouse aorta. The overexpression of C/EBPβ in A10 cells increased bone runt-related transcription factor 2 ( Runx2 ), alkaline phosphatase ( ALP ), and osteopontin ( OPN ) mRNA in the presence of Pi, as well as potentiating the Pi-induced increase in calcium contents. The Runx2 expression was increased by C/EBPβ through Runx2 P2 promotor. Our results suggest that a Pi-induced increase in C/EBPβ is a critical step in vascular calcification.

Mitotic inhibitors are drugs commonly used in chemotherapy, but their nonspecific and indiscriminate distribution throughout the body after intravenous administration can lead to serious side effects, particularly on the cardiovascular system. In this context, our investigation into the mechanism of the cytotoxic effects on endothelial cells of mitotic inhibitors widely used in cancer treatment, such as paclitaxel (also known as Taxol) and Vinca alkaloids, holds significant practical implications. Understanding these mechanisms can lead to more targeted and less harmful cancer treatments. Human aorta endothelial cells (HAECs) were incubated with selected mitotic inhibitors in a wide range of concentrations close to those in human plasma during anticancer therapy. The analysis of single cells imaged by Raman spectroscopy allowed for visualization of the nuclear, cytoplasmic, and perinuclear areas to assess biochemical changes induced by the drug's action. The results showed significant changes in the morphology and molecular composition of the nucleus. Moreover, an effect of a given drug on the cytoplasm was observed, which can be related to its mechanism of action (MoA). Raman data supported by fluorescence microscopy measurements identified unique changes in DNA form and proteins and revealed drug-induced inflammation of endothelial cells. The primary goal of mitotic inhibitors is based on the impairment of tubulin formation and the inhibition of the mitosis process. While all three drugs affect microtubules and disrupt cell division, they do so through different MoA, i.e., Vinca alkaloids inhibit microtubule formation, whereas paclitaxel stabilizes microtubules. To sum up, the work shows how a specific drug can interact with endothelial cells., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships that may be considered potential competing interests: Katarzyna Majzner reports that National Science Centre Poland provided financial support. Professor Malgorzata Baranska, who co-authored the paper, also serves as an editor at SAA. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heparan sulfate (HS) is the most abundant glycosaminoglycan on the vascular endothelium and can regulate endothelial cell morphology and function in response to mechanical stimuli. This study investigated endothelial HS response to an inflammatory stimulus under static and arterial shear stress conditions. Human aortic endothelial cells (HAECs) under static conditions expressed significantly higher HS when treated with an inflammatory stimulus compared to untreated controls. HAECs exposed to an inflammatory stimulus after being conditioned with 10 dyn/cm 2 of shear stress for 24 h did not express significantly higher HS compared to untreated controls under flow. To investigate the mechanism underlying this differential endothelial HS expression in response to an inflammatory stimulus under static and shear stress conditions, we hypothesized a shear dependent increase in AMP dependent protein kinase (AMPK) was regulating HS response to the inflammatory stimulus. AMPK inhibition using compound C decreased HAEC HS expression in response to inflammatory stimulus under arterial shear stress, revealing AMPK as a regulator of HS expression. Further investigation is needed to elucidate the mechanistic pathways underlying the interactions between HS and AMPK expression in endothelial cells and how they regulate HAEC inflammatory response., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Increased dietary salt intake is a well-established risk factor for hypertension and related cardiovascular diseases, involving complex vascular remodeling processes. However, the specific role of hypoxia-inducible factor-1α (HIF-1α) in vascular pathophysiology under high-salt conditions remains poorly understood. This study investigates the role of HIF-1α in high-salt-induced vascular remodeling using human aortic vascular smooth muscle cells (HA-VSMCs) cultured in vitro. HA-VSMCs were divided into three groups: high-salt with HIF-1α knockdown (shHIF-1α + HS), negative control (shcontrol), and high-salt (HS). Cell viability, migration, gene expression, and protein levels were evaluated. High-salt conditions significantly increased mRNA expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), angiotensin II type 1 receptor (AT1R), collagen I, and collagen III (p < 0.0001). HIF-1α knockdown partially attenuated these increases, particularly for α-SMA, SM22, and AT1R (p < 0.01). At the protein level, high-salt exposure markedly elevated expression of collagen III, HIF-1α, osteopontin (OPN), and angiotensin II (Ang II) (p < 0.0001). HIF-1α knockdown significantly reduced the high-salt-induced increases in collagen III and HIF-1α protein levels (p < 0.001) but had a limited effect on OPN and Ang II upregulation. Interestingly, SM22 protein expression was significantly decreased under high-salt conditions (p < 0.0001), an effect partially reversed by HIF-1α knockdown (p < 0.0001). These findings demonstrate that high-salt conditions induce complex changes in gene and protein expression in HA-VSMCs, with HIF-1α playing a crucial role in mediating many of these alterations. The study highlights the differential effects of HIF-1α on various markers of vascular remodeling and suggests that HIF-1α may be a potential therapeutic target for mitigating salt-induced vascular pathology. Further research is warranted to elucidate the mechanisms underlying the HIF-1α-dependent and -independent effects observed in this study., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Objective: To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells., Methods: The thoracic and abdominal aortas isolated from 2- to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes. The primary cells were further cultured to 80%-90% confluence before passaging. The morphology and growth characteristics of the cells were observed under a microscope, and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry. Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation., Results: After 3 days of culture, a small number of spindle, star-shaped or polygonal cells migrated out from the peripheral of the vascular segments. At 5-6 days, island-like cell clusters occurred and the cells began to proliferate rapidly. The cell clusters expanded radially and showed signs of cell cloning. At 7-8 days, the cells fused into sheets and displayed a vortex-like distribution. The cells in the third passage presented with a uniform morphology, showing a typical fibroblast-like arrangement. Flow cytometry showed that the cells expressed predominantly CD44 (80.3%), CD73 (62.2%) and CD90 (46.8%) with low expressions of CD34 (1.1%), CD45 (0.2%) and CD11b/c (0.2%). Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro ., Conclusion: Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

Wall shear stress (WSS) is a critical factor in vascular biology, and both high and low WSS are implicated in atherosclerosis. Fibronectin (FN) is a key extracellular matrix protein that plays an important role in cell activities. Under high shear stress, plasma FN undergoes fibrillogenesis; however, its behavior under low shear stress remains unclear. This study aimed to investigate the formation of in vitro cell-free fibrillar FN (FFN) under low shear rate conditions and its effect on bovine aortic endothelial cell behavior. FN (500 µ g ml -1 ) was perfused through slide chambers at three flow rates (0.16 ml h -1 , 0.25 ml h -1 , and 0.48 ml h -1 ), corresponding to low shear rates of 0.35 s -1 , 0.55 s -1 , and 1.05 s -1 , respectively, for 4 h at room temperature. The formed FN matrices were observed using fluorescence microscopy and scanning electron microscopy. Under low shear rates, distinct FN matrix structures were observed. FFN0.48 formed immense fibrils with smooth surfaces, FFN0.25 formed a matrix with a rough surface, and FFN16 exhibited nodular structures. FFN0.25 supported cell activities to a greater extent than native FN and other FFN surfaces. Our study suggests that abnormally low shear conditions impact FN structure and function and enhance the understanding of FN fibrillogenesis in vascular biology, particularly in atherosclerosis., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Vascular aging is associated with the development of cardiovascular complications, in which endothelial cell senescence (ES) may play a critical role. Nitric oxide (NO) prevents human ES through inhibition of oxidative stress, and inflammatory signaling by mechanisms yet to be elucidated. Endothelial cells undergo an irreversible growth arrest and alter their functional state after a finite number of divisions, a phenomenon called replicative senescence. We assessed the contribution of NO during replicative senescence of human aortic (HAEC) and coronary (CAEC) endothelial cells, in which accumulation of the senescence marker SA-β-Gal was quantified by β-galactosidase staining on cultured cells. We found a negative correlation in passaged cell cultures from P0 to P12, between a reduction in NO production with increased ES and the formation of reactive oxygen (ROS) and nitrogen (ONOO - ) species, indicative of oxidative and nitrosative stress. The effect of ES was evidenced by reduced expression of endothelial Nitric Oxide Synthase (eNOS), Interleukin Linked Kinase (ILK), and Heat shock protein 90 (Hsp90), alongside a significant increase in the BH2/BH4 ratio, inducing the uncoupling of eNOS, favoring the production of superoxide and peroxynitrite species, and fostering an inflammatory environment, as confirmed by the levels of Cyclophilin A (CypA) and its receptor Extracellular Matrix Metalloprotease Inducer (EMMPRIN). NO prevents ES by preventing the uncoupling of eNOS, in which oxidation of BH4, which plays a key role in eNOS producing NO, may play a critical role in launching the release of free radical species, triggering an aging-related inflammatory response.

Drugs that act on α-adrenoceptors may contain morpholine and pyrimidinone heterocycles. The aim of this study was to synthesize a series of pyrimidinones (S6a-e and S8) and characterize their α-adrenoceptor activity. Cytotoxicity assays (MTT and LDH) were performed in A7r5 and HUVECs. Concentration-effect curves to phenylephrine (Phe) were performed in rat aortic rings in the presence of compounds S6a-e and S8 or vehicle. Nitric oxide (NO) production and NO stable metabolic products, nitrite and nitrate, expressed as total nitrogen oxides (NOx) were assessed in HUVECs by confocal microscopy with the DAF-2DA probe and by the Griess reaction, respectively. Molecular docking simulations were performed using the 6a compound and α 2A -adrenoceptor. In the evaluated conditions, the percentage of viable cells and the release of LDH were similar between control cells and cells exposed to the tested pyrimidinones. S6d, S6e, S8, and the positive control prazosin (but not S6a, S6b, and S6c) decreased Phe-induced contractions in endothelium-denuded aortic rings. S6a, S6b, and S6c decreased Phe-induced contractions in endothelium-intact aortic rings. The effect of S6a was abolished by L-NAME. NO production and NOx levels were inhibited in the presence of the α 2 receptor antagonist yohimbine and the NOS inhibitor L-NAME. The 6a docking simulation estimated that the mean binding free energy of the compound was lower than the estimated value for yohimbine. These data suggest that S6d, S6e, and S8 may be α 1 -adrenoceptor antagonists while S6a acts as an agonist of α 2 -adrenoceptors., Competing Interests: Declaration of competing interest The authors of the manuscript entitled “New morpholine-containing pyrimidinones act on alpha-adrenergic receptors” declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Vascular smooth muscle cell (VSMC) phenotype switch and dysfunctions have been reported to participate in aortic dissection (AD) progression. This study was aimed to investigate the role of angiopoietin-like 4 (ANGPTL4) in regulating VSMCs phenotype switch., Materials and Methods: Key genes were analyzed in AD using public datasets, and it was found that the central differential gene ANGPTL4 was up-regulated in AD. The KEGG signaling pathway annotation was performed to validate the associated pathways, and the expression of ANGPTL4 was verified using multiple datasets and clinical samples. Furthermore, the specific functions of ANGPTL4 on platelet-derived growth factor-BB (PDGF-BB)-treated human aortic smooth muscle cell (HASMC) phenotypes were investigated. The dynamic effects of ANGPTL4 and core signaling antagonists on HASMC phenotypes were examined., Results: Hub gene ANGPTL4 was significantly up-regulated in AD. ANGPTL4 was linked to the PI3K/Akt signaling, angiogenesis, and neovascularization and remodeling. ANGPTL4 overexpression further enhanced PDGF-BB effects on HASMC phenotypes, including promoted cell viability and migration, decreased contractile VSMC markers α-SMA and SM22α, elevated ECM degradation markers MMP-2 and MMP-9, and promoted phosphorylation of PI3K and Akt. ANGPTL4 knockdown partially abolished PDGF-BB-induced contractile/synthetic VSMCs imbalance and HASMC dysfunctions. Furthermore, in ANGPTL4-overexpressing HASMCs pre-treated with PDGF-BB, the PI3K/Akt signaling inhibitor LY294002 also partially eliminated the effects caused by the PDGF-BB treatment and ANGPTL4 overexpression., Conclusions: ANGPTL4 is significantly up-regulated in AD. ANGPTL4 overexpression further enhanced PDGF-BB effects on HASMC phenotype switch and dysfunctions, which might be involved in the PI3K/Akt signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

An intricate reciprocal relationship exists between adherent synthetic cells and their extracellular matrix (ECM). These cells deposit, organize, and degrade the ECM, which in turn influences cell phenotype via responses that include sensitivity to changes in the mechanical state that arises from changes in external loading. Collagen-based tissue equivalents are commonly used as simple but revealing model systems to study cell-matrix interactions. Nevertheless, few quantitative studies report changes in the forces that the cells establish and maintain in such gels under dynamic loading. Moreover, most prior studies have been limited to uniaxial experiments despite many soft tissues, including arteries, experiencing multiaxial loading in vivo. To begin to close this gap, we use a custom biaxial bioreactor to subject collagen gels seeded with primary aortic smooth muscle cells to different biaxial loading conditions. These conditions include cyclic loading with different amplitudes as well as different mechanical constraints at the boundaries of a cruciform sample. Irrespective of loading amplitude and boundary condition, similar mean steady-state biaxial forces emerged across all tests. Additionally, stiffness-force relationships assessed via intermittent equibiaxial force-extension tests showed remarkable similarity for ranges of forces to which the cells adapted during periods of cyclic loading. Taken together, these findings are consistent with a load-mediated homeostatic response by vascular smooth muscle cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Etravirine (ETV) is an antiretroviral agent that belongs to the class of non-nucleoside reverse transcriptase inhibitors. This study explores the uptake and distribution of ETV in human aortic endothelial cells (HAECs) using Raman spectroscopy combined with chemometrics. The distinctive chemical structure of ETV facilitates tracking of its uptake by observing the Raman band at 2225 cm -1 in the Raman-silent region. The perinuclear distribution pattern in HAECs depends on drug concentration and incubation time. The uptake of ETV is observed within 5 minutes at a concentration of 10 μM, as evidenced by Raman images. Lower ETV concentrations, reflective of those found in human plasma, are detectable in HAECs by applying chemometric methods to Raman spectra from the perinuclear region. The ETV accumulation process is crucial in advancing our understanding of the drug's impact on biochemical alterations within endothelial cells. Additionally, ETV emerges as a promising Raman reporter for marking subcellular compartments, leveraging the 2225 cm -1 band in the cellular Raman silent region. This research contributes valuable insights into the behavior of ETV at the subcellular level, shedding light on its potential applications and impact on subcellular dynamics.

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX 3 CR1 + and CSF1R + source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally., (© 2024. The Author(s).)

Background: The extracellular vesicles (EVs) secreted by adipose tissue-derived stromal cells (ASC) are microenvironment modulators in tissue regeneration by releasing their molecular cargo, including miRNAs. However, the influence of ASC-derived extracellular vesicles (ASC-EVs) on endothelial cells (ECs) and vascularisation is poorly understood. The present study aimed to determine the pro-angiogenic effects of ASC-EVs and explore their miRNA profile., Methods: EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24 h. ASC-EVs' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®)., Results: ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs., Conclusion: Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels., (© 2024. The Author(s).)

Adult blood cells are produced in the bone marrow by hematopoietic stem cells (HSCs), the origin of which can be traced back to fetal developmental stages. Indeed, during mouse development, at days 10-11 of gestation, the aorta-gonad-mesonephros (AGM) region is a primary site of HSC production, with characteristic cell clusters related to stem cell genesis observed in the dorsal aorta. Similar clusters linked with hematopoiesis are also observed in the other sites such as the yolk sac and placenta. In this review, I outline the formation and function of these clusters, focusing on the well-characterized intra-aortic hematopoietic clusters (IAHCs)., Competing Interests: Conflicts of Interest Disclosure The authors have nothing to disclose., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Vascular smooth muscle cell (VSMC) dedifferentiation contributes substantively to vascular disease. VSMCs spontaneously release low levels of ATP that modulate vessel contractility, but it is unclear if autocrine ATP signaling in VSMCs is critical to the maintenance of the VSMC contractile phenotype., Methods: We used pharmacological inhibitors to block ATP release in human aortic smooth muscle cells (HASMCs) for studying changes in VSMC differentiation marker gene expression. We employed RNA interference and generated mice with SMC-specific inducible deletion of the P2Y 2 receptor (P2Y 2 R) gene to evaluate resulting phenotypic alterations., Results: HASMCs constitutively release low levels of ATP that when blocked results in a significant decrease in VSMC differentiation marker gene expression, including smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), SM-22α and calponin. Basal release of ATP represses transcriptional activation of the Krüppel-Like Factor 4 (KFL4) thereby preventing platelet-derived growth factor-BB (PDGF-BB) from inhibiting expression of SMC contractile phenotype markers. SMC-restricted conditional deletion of P2Y 2 R evoked dedifferentiation characterized by decreases in aortic contractility and contractile phenotype markers expression. This loss was accompanied by a transition to the synthetic phenotype with the acquisition of extracellular matrix (ECM) proteins characteristic of dedifferentiation, such as osteopontin and vimentin., Conclusions: Our data establish the first direct evidence that an autocrine ATP release mechanism maintains SMC cytoskeletal protein expression by inhibiting VSMCs from transitioning to a synthetic phenotype, and further demonstrate that activation of the P2Y 2 R by basally released ATP is required for maintenance of the differentiated VSMC phenotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Current knowledge indicates that the consumption of isoflavone-rich foodstuffs can have a beneficial impact on cardiovascular health. To what extent these isoflavones act as the main actors of that benefit is less clear. Genistein (GEN), daidzein (DAZ), and the DAZ-derived microbial metabolite equol (Eq) exhibit antiangiogenic effects in vitro , but their low bloodstream concentrations make it difficult to rationalize the in vivo effects. Their derived phase-II metabolites (glucuronides and sulfates) are major metabolites found in plasma, but their role as antiangiogenic molecules remains unexplored. We aimed here to first assess the anti-angiogenic activities of the main circulating isoflavone metabolites (glucuronides and sulfates) and compare them with their corresponding free forms at physiological concentrations (0.1-10 μM). The effects of the conjugated vs. free forms on tubulogenesis, cell migration, and VEGF-induced signalling were investigated in primary human aortic endothelial cells (HAECs). While ( R , S )-equol 7-β-D-glucuronide (Eq 7-glur) exerted dose-dependent inhibition of tubulogenesis and endothelial migration comparable to that exerted by the free forms (GEN, DAZ, and Eq), the rest of the phase-II conjugates exhibited no significant effects. The underlying molecular mechanisms were independent of the bFGF but related to the modulation of the VEGF pathway. Besides, the observed dissimilar cellular metabolism (conjugation/deconjugation) places the phase-II metabolites as precursors of the free forms; however, the question of whether this metabolism impacts their biological activity requires additional studies. These new insights suggest that isoflavones and their circulating metabolites, including Eq 7-glur, may be involved in cardiovascular health ( e.g. , targeting angiogenesis).

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca 2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca 2+ dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study aims to examine the effect of salidroside(SAL) on the phenotypic switching of human aortic smooth muscle cells(HASMC) induced by the platelet-derived growth factor-BB(PDGF-BB) and investigate the pharmacological mechanism. Firstly, the safe concentration of SAL was screened by the lactate dehydrogenase release assay. HASMC were divided into control, model, and SAL groups, and the cells in other groups except the control group were treated with PDGF-BB for the modeling of phenotypic switching. Cell proliferation and migration were detected by the cell-counting kit(CCK-8) assay and Transwell assay, respectively. The cytoskeletal structure was observed by F-actin staining with fluorescently labeled phalloidine. The protein levels of proliferating cell nuclear antigen(PCNA), migration-related protein matrix metalloprotein 9(MMP-9), fibronectin, α-smooth muscle actin(α-SMA), and osteopontin(OPN) were determined by Western blot. To further investigate the pharmacological mechanism of SAL, this study determined the expression of protein kinase B(Akt) and mammalian target of rapamycin(mTOR), as well as the upstream proteins phosphatase and tensin homologue(PTEN) and platelet-derived growth factor receptor β(PDGFR-β) and the downstream protein hypoxia-inducible factor-1α(HIF-1α) of the Akt/mTOR signaling pathway. The results showed that the HASMCs in the model group presented significantly increased proliferation and migration, the switching from a contractile phenotype to a secretory phenotype, and cytoskeletal disarrangement. Compared with the model group, SAL weakened the proliferation and migration of HASMC, promoted the expression of α-SMA(a contractile phenotype marker), inhibited the expression of OPN(a secretory phenotype marker), and repaired the cytoskeletal disarrangement. Furthermore, compared with the control group, the modeling up-regulated the levels of phosphorylated Akt and mTOR and the relative expression of PTEN, HIF-1α, and PDGFR-β. Compared with the model group, SAL down-regulated the protein levels of phosphorylated Akt and mTOR, PTEN, PDGFR-β, and HIF-1α. In conclusion, SAL exerts a protective effect on the HASMCs exposed to PDGF-BB by regulating the PDGFR-β/Akt/mTOR/HIF-1α signaling pathway.

The effect of endothelial cells' exposure to dibutyl phthalate (DBP) on monocyte adhesion is largely unknown. We evaluated monocyte adhesion to DBP-exposed endothelial cells by combining three approaches: short-term exposure (24 h) of EA.hy926 cells to 10 -6 , 10 -5 , and 10 -4  M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10 -9 , 10 -8 , and 10 -7  M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. Monocyte adhesion to human EA.hy926 and rat aortic endothelial cells, expression of selected cellular adhesion molecules and chemokines, and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) were analyzed. We observed increased monocyte adhesion to DBP-exposed EA.hy926 cells in vitro and to rat aortic endothelium ex vivo. ERK1/2 inhibitor prevented monocyte adhesion to DBP-exposed EA.hy926 cells in short-term exposure experiments. Increased ERK1/2 phosphorylation in rat aortic endothelium and transient decrease in ERK1/2 activation following long-term exposure of EA.hy926 cells to DBP were also observed. In summary, exposure of endothelial cells to DBP promotes monocyte adhesion, thus suggesting a possible role for this phthalate in the development of atherosclerosis. ERK1/2 signaling could be the mediator of monocyte adhesion to DBP-exposed endothelial cells, but only after short-term high-level exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background and Purpose: Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined., Experimental Approach: Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation., Key Results: On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation., Conclusions and Implications: In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

To provide a theoretical basis for the prevention and treatment of atherosclerosis (AS), the current study aimed to investigate the mechanism underlying the effect of homocysteine (Hcy) on regulating the proliferation, migration and phenotypic transformation of vascular smooth muscle cells (VSMC) via sirtuin-1 (SIRT1)/signal transducer and activator of transcription 3 (STAT3) through Nedd4-like E3 ubiquitin-protein ligase WWP2 (WWP2). Here, Based on the establishment of ApoE-/- mouse models of high Hcy As and the model of Hcy stimulation of VSMC in vitro to observe the interaction between WWP2 and STAT3 and its effect on the proliferation, migration, and phenotypic transformation of Hcy-induced VSMC, which has not been previously reported. This study revealed that WWP2 could promote the proliferation, migration, and phenotype switch of Hcy-induced VSMC by up-regulating the phosphorylation of SIRT1/STAT3 signaling. Furthermore, Hcy might up-regulate WWP2 expression by inhibiting histone H3K27me3 expression through up-regulated UTX. These data suggest that WWP2 is a novel and important regulator of Hcy-induced VSMC proliferation, migration, and phenotypic transformation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Senolytics are drugs that specifically target senescent cells. Flavonoids such as quercetin and fisetin possess selective senolytic activities. This study aims to investigate if chalcones exhibit anti-senescence activities. Anti-senescence effect of 11 chalcone derivatives on the replicative senescence human aortic endothelial cells (HAEC) and human fetal lung fibroblasts (IMR90) was evaluated. Compound 2 (4-methoxychalcone) and compound 4 (4-bromo-4'-methoxychalcone) demonstrated increased cytotoxicity in senescent HAEC compared to young HAEC, with significant differences on IC 50 values. Their anti-senescence effects on HAEC exceeded fisetin. Higher selectivity of compound 4 toward HAEC over IMR90 could be attributed to 4-methoxy (4-OMe) substitution at ring A (R1). Chalcone derivatives have potentials as senolytics in mitigating replicative senescence, warranting further research and development on chalcones as anti-senescent agent.

Recent studies have focused on the contribution of vascular endothelial transient receptor potential vanilloid 4 (TRPV4) channels to hypertension. However, in hypertension, TRPV4 channels in vascular smooth muscle remain unexplored. In the present study, we performed wire myograph experiments in isolated aortas from endothelial cell specific TRPV4 channel knockout (TRPV4 EC -/- ) mice to demonstrate that GSK1016790A (a specific TRPV4 channel agonist) triggered aortic smooth muscle-dependent contractions from mice on a normal-salt diet, and the contractions were enhanced in high-salt diet (HSD) mice. Intracellular Ca 2+ concentration ([Ca 2+ ] i ) and Ca 2+ imaging assays showed that TRPV4-induced [Ca 2+ ] i was significantly higher in aortic smooth muscle cells (ASMCs) from HSD-induced hypertensive mice, and application of an inositol trisphosphate receptor (IP 3 R) inhibitor markedly attenuated TRPV4-induced [Ca 2+ ] i . IP 3 R2 expression was enhanced in ASMCs from HSD-induced hypertensive mice and the contractile response induced by TRPV4 was inhibited by the IP 3 R inhibitor. Whole-transcriptome analysis by RNA-seq and western blot assays revealed the involvement of interferon regulatory factor 7 (IRF7) in TRPV4-IRF7-IP 3 R2 signaling in HSD-induced hypertension. These results suggested that TRPV4 channels regulate smooth muscle-dependent contractions in high salt-induced hypertension, and this contraction involves increased [Ca 2+ ] i , IP 3 R2, and IRF7 activity. Our study revealed a considerable effect of TRPV4 channels in smooth muscle-dependent contraction in mice during high-salt induced hypertension., (© 2023. The Author(s), under exclusive licence to The Japanese Society of Hypertension.)

Intimal thickening caused by the excessive multiplication of vascular smooth muscle cells (VSMCs) is the pathological process central to cardiovascular diseases, including restenosis. In response to vascular injury, VSMCs would undergo phenotypic switching from a fully differentiated, low proliferative rate phenotype to a more pro-proliferative, promigratory, and incompletely-differentiated state. The lack of a full understanding of the molecular pathways coupling the vascular injury stimuli to VSMCs phenotype switching largely limits the development of medical therapies for treating intima hyperplasia-related diseases. The role of signal transducers and activators of transcription 6 (STAT6) in modulating the proliferation and differentiation of various cell types, especially macrophage, has been well investigated, but little is known about its pathophysiological role and target genes in restenosis after vascular injury. In the present work, Stat6 -/- mice were observed to exhibit less severe intimal hyperplasia compared with Stat6 +/+ mice after carotid injury. The expression of STAT6 was upregulated in VSMCs located in the injured vascular walls. STAT6 deletion leads to decreased proliferation and migration of VSMCs while STAT6 overexpression enhances the proliferation and migration of VSMCs companies with reduced expression of VSMCs marker genes and organized stress fibers. The effect of STAT6 in mouse VSMCs was conserved in human aortic SMCs. RNA-deep-sequencing and experiments verification revealed LncRNA C7orf69/LOC100996318-miR-370-3p/FOXO1-ER stress signaling as the downstream network mediating the pro-dedifferentiation effect of STAT6 in VSMCs. These findings broaden our understanding of vascular pathological molecules and throw a beam of light on the therapy of a variety of proliferative vascular diseases., (© 2023 International Federation of Cell Biology.)

Context: Vascular calcification is a major complication of chronic renal failure, which has been identified as an active process partly driven by osteogenic transition of vascular smooth muscle cells (VSMCs). Aspirin could prevent cardiomyocyte damage by inducing heat shock response., Objective: This study investigates the effect of aspirin on alleviating VSMC calcification., Materials and Methods: An in vitro VSMC calcification model was established by 10-day calcification induction in osteogenic medium. VSMCs were grouped as following: control group (normal medium), calcified group (osteogenic medium) and treated group (osteogenic medium with 1 or 4 mmol/L aspirin). VSMC calcification was evaluated by calcified nodules formation, intracellular calcium concentration and osteoblastic marker (OPN and Runx2) expression., Results: After 10-day culture, the intracellular calcium concentration in calcified group was significantly higher than that in control group (1.16 ± 0.04 vs. 0.14 ± 0.01 μg/mg, p  < 0.01), but significantly reduced in 1 mmol/L aspirin treated group (0.74 ± 0.05 μg/mg, p  < 0.01), and 4 mmol/L aspirin treated group (0.93 ± 0.03 μg/mg, p  < 0.01). The elevated expression of OPN and Runx2 induced by osteogenic medium was significantly relieved after 1 or 4 mmol/L aspirin treatment. The expression of HSF1, HSP70 and HSP90 was decreased in calcification-induced VSMCs, but significantly increased after treatment of aspirin. Furthermore, inhibition of HSP70 (or HSP90) by small-molecule inhibitor or small interfering RNA could partially abolish the anti-calcification effect of aspirin, proved by the changes of intracellular calcium concentration and osteoblastic marker expression., Discussion and Conclusions: Aspirin could relieve the calcification of VSMCs partially through HSP70- or HSP90-mediated heat shock response. These findings expanded the understanding of aspirin pharmacology, and imply that local induction expression of HSPs might be a potential therapeutic strategy for the prevention and therapy of vascular calcification.

Tissue-resident macrophages play essential functions in the maintenance of tissue homeostasis and repair. Recently, the endocardium has been reported as a de novo hemogenic site for the contribution of hematopoietic cells, including cardiac macrophages, during embryogenesis. These observations challenge the current consensus that hematopoiesis originates from the hemogenic endothelium within the yolk sac and dorsal aorta. Whether the developing endocardium has such a hemogenic potential requires further investigation. Here, we generated new genetic tools to trace endocardial cells and reassessed their potential contribution to hematopoietic cells in the developing heart. Fate-mapping analyses revealed that the endocardium contributed minimally to cardiac macrophages and circulating blood cells. Instead, cardiac macrophages were mainly derived from the endothelium during primitive/transient definitive (yolk sac) and definitive (dorsal aorta) hematopoiesis. Our findings refute the concept of endocardial hematopoiesis, suggesting that the developing endocardium gives rise minimally to hematopoietic cells, including cardiac macrophages., (© 2022 liu et al.)

Aortic dissection (AD), also known as aortic dissecting aneurysm, is one of the most common and dangerous cardiovascular diseases with high morbidity and mortality. This study was aimed to investigate the functional role of long non-coding RNA Hypoxia-inducible factor 1 alpha-antisense RNA 2 (lncRNA HIF1A-AS2) in AD. An in vitro model of AD was established by platelet-derived growth factor-BB (PDGF-BB)-mediated human aortic Smooth Muscle Cells (SMCs). HIF1A-AS2 expression in human AD tissues was determined by quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) assays, followed by investigation of biological roles of HIF1A-AS2 in AD development by Cell Counting Kit-8 (CCK-8), immunofluorescence, and transwell assays. Additionally, the correlation between HIF1A-AS2, miR-33b, and high mobility group AT-hook2 (HMGA2) were identified by RNA immunoprecipitation (RIP), RNA pull-down and luciferase reporter assays. Results showed that HIF1A-AS2 was obviously increased, while the contractile-phenotype markers of vascular SMCs were significantly decreased in human AD tissues, when compared to normal tissues. Inhibition of HIF1A-AS2 attenuated SMCs proliferation and migration, whereas enhanced the phenotypic switch under the stimulation of PDGF-BB. Results from RIP, RNA pull-down and luciferase reporter assays demonstrated that miR-33b directly bound with HIF1A-AS2, and HIF1A-AS2 silencing suppressed the expression of HMGA2, which was induced by miR-33b inhibitor. In conclusion, knockdown of HIF1A-AS2 suppressed the proliferation and migration, while promoted the phenotypic switching of SMCs through miR-33b/HMGA2 axis, which laid a theoretical foundation for understanding the development of AD and shed light on a potential target for AD treatment.

Nitric oxide (NO) binds soluble guanylyl cyclase β (sGCβ) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCβ in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCα and 50% loss of sGCβ protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCβ expression in SMC. NEW & NOTEWORTHY One of the key mechanisms of vascular smooth muscle cell (SMC) dilation occurs through nitric oxide (NO)-dependent induction of soluble guanylyl cyclase (sGC) by means of its β-subunit. Herein, we are the first to identify Forkhead box subclass O protein 4 (FoxO4) as a key transcriptional regulator of GUCY1B3 expression, which codes for sGCβ protein in human and animal SMCs. This discovery will likely have important implications for the future usage of antihypertensive and vasodilatory therapies which target NO production, sGC, or FoxO transcription factors.

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Abdominal aortic aneurysm (AAA) has been associated with the dysfunction of vascular smooth muscle cells (VSMCs) and extracellular matrix (ECM) remodelling. Runt-related transcription factor 3 (RUNX3) has been reported to be up-regulated in aneurysmal aorta samples compared with normal aorta. However, its function in VSMCs and the mechanism of function remains unknown. Therefore, our study aimed to investigate the role of RUNX3 in ECM remodelling and VSMC function, and further explore the underlying mechanism. Our results verified that RUNX3 was increased in aortic samples of AAA compared with healthy controls. Overexpression vectors of RUNX3 (ov-RUNX3) and siRNA of RUNX3 (si-RUNX3) were transfected into Human aortic smooth muscle cells (HAoSMCs). The results indicated that ov-RUNX3 promoted cell proliferation, migration, and MMP-2/3/9 secretion, and suppressed TIMP-1, collagen I/III, SM22, MYH11 and CNN1 expression in HAoSMCs. The silencing of RUNX3 has the opposite effect. Furthermore, we found that RUNX3 targets TGF-β1 and suppressed its transcription. The silencing of TGF-β1 increased cell proliferation, migration and MMP-2/3/9 expression, and inhibited TIMP-1, Collagen I/III, SM22, MYH11 and CNN1 expression. In addition, TGF-β1 reversed the effect of RUNX3 overexpression on HAoSMCs. Hence, our study indicated that RUNX3 promotes cell proliferation, migration, and ECM remodelling through suppressing TGF-β1., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) during development in a process called endothelial-to-hematopoietic transition. The factors and signals that control HSPC fate decisions from the hemogenic endothelium are not fully understood. We found that Vegfc has a role in HSPC emergence from the zebrafish DA. Using time-lapse live imaging, we show that some HSPCs in the DA of vegfc loss-of-function embryos display altered cellular behavior. Instead of typical budding from the DA, emergent HSPCs exhibit crawling behavior similar to myeloid cells. This was confirmed by increased myeloid cell marker expression in the ventral wall of the DA and the caudal hematopoietic tissue. This increase in myeloid cells corresponded with a decrease in HSPCs that persisted into larval stages. Together, our data suggest that Vegfc regulates HSPC emergence in the hemogenic endothelium, in part by suppressing a myeloid cell fate. Our study provides a potential signal for modulation of HSPC fate in stem cell differentiation protocols., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Emerging evidence has shown that nonalcoholic fatty liver disease (NAFLD) may be both a consequence and a cause of hypertension. Recent studies have demonstrated that phosphodiesterase 4 (PDE4)-cAMP signaling represents a pathway relevant to the pathophysiology of metabolic disorders. This study aims to investigate the impact and the underlying mechanism of PDE4 in the pathogenesis of NAFLD and its associated hypertension. Here we demonstrated that high-fat-diet (HFD) fed mice developed NAFLD and hypertension, with an associated increase in hepatic PDE4D expression, which can be prevented and even reversed by PDE4 inhibitor roflumilast. Furthermore, we demonstrated that hepatic overexpression of PDE4D drove significant hepatic steatosis and elevation of blood pressure. Mechanistically, PDE4D activated fatty acid translocase CD36 signaling which facilitates hepatic lipid deposition, resulting in TGF-β1 production by hepatocytes and excessive TGF-β1 signaling in vessels and consequent hypertension. Specific silencing of TGF-β1 in hepatocytes by siRNA using poly (β-amino ester) nanoparticles significantly normalized hepatic PDE4D overexpression-activated TGF-β1 signaling in vessels and hypertension. Together, the conclusions indicated that PDE4D plays an important role in the pathogenesis of NAFLD and associated hypertension via activation of CD36-TGF-β1 signaling in the liver. PDE4 inhibitor such as roflumilast, which is clinically approved for chronic obstructive pulmonary disease (COPD) treatment, has the potential to be used as a preventive or therapeutic drug against NAFLD and associated hypertension in the future., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.

SARS-CoV-2 infection can cause compromised respiratory function and thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. Theoretically, diminished enzymatic activity of ACE2 may result in increased concentrations of pro-inflammatory molecules, angiotensin II, and Bradykinin, contributing to SARS-CoV-2 pathology. Using immunofluorescence microscopy of lung tissues from uninfected, and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in human pulmonary alveolar epithelial cells and significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses of primary human cells, indicated that ACE2 is readily detected in pulmonary alveolar epithelial and aortic endothelial cells. Exposure of these cells to spike protein of SARS-CoV-2 was sufficient to reduce ACE2 expression. Moreover, exposure of endothelial cells to spike protein-induced dysfunction, caspase activation, and apoptosis. Exposure of endothelial cells to bradykinin caused calcium signaling and endothelial dysfunction (increased expression of von Willibrand Factor and decreased expression of Krüppel-like Factor 2) but did not adversely affect viability in primary human aortic endothelial cells. Computer-assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2), suggested a potential role for aspirin as a BK antagonist. When tested in our in vitro model, we found evidence that aspirin can blunt cell signaling and endothelial dysfunction caused by bradykinin in these cells. Interference with interactions of spike protein or bradykinin with endothelial cells may serve as an important strategy to stabilize microvascular homeostasis in COVID-19 disease. IMPORTANCE SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. It is thought that reduced ACE2 enzymatic activity can contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions that could limit microvascular events associated with SARS-CoV-2 infection.

(1) Background: The anthocyanin delphinidin exhibits anti-angiogenic properties both in in vitro and in vivo angiogenesis models. However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (sEV) properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells (HAoECs). (2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide (NO) production and Matrigel assay were evaluated in HAoECs. (3) Results: Delphinidine, 3-O-β-rutinoside and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs. sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation, 10-fold for endothelial NO production and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions: sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization.

The cardiovascular is a system that contains extremely complex mechanical factors, in which the circulatory flow of blood has rich mechanical laws. Many studies have revealed that mechanical factors play a very important role in the process of revascularization. Hence, it is essential to investigate the mechanical factors in the process of revascularization in depth. A cyclic tensile strain (CTS) was applied to human aortic smooth muscle cells (HASMCs) at a frequency of 1 Hz and amplitudes of 5%, 10% and 15%, respectively. SmallRNA-seq was used to identify differentially expressed miRNAs (DE-miRNAs) responding to CTS in HASMCs. Starbase database predicted the target genes of DE-miRNAs. Metascape was applied for GO and KEGG pathway enrichment analysis and protein-protein interaction network construction. The proliferation and migration of CTS-treated HASMCs were significantly enhanced, and apoptosis were significantly reduced compared to the control group. SmallRNA-seq results demonstrated that 55, 16 and 16 DE-miRNAs were present in 5%, 10% and 15% CTS-treated HASMCs, respectively. Compared to controls, with miR-26a-2-3p and miR-187-3p being the intersection of these DE-miRNAs. Starbase database identified 189 common target genes for miR-26a-2-3p and miR-187-3p. Common target genes are mainly enriched in the basolateral plasma membrane and endocytosis. Further, in vitro experiments exhibited that CTS upregulated miR-187-3p expression, and miR-187-3p enhanced the proliferation and migration of HASMCs and reduced their apoptosis. It is suggested that miR-187-3p may be an important target for CTS participate in the process of cardiovascular disease.[Figure: see text].

Atherosclerosis (AS) is one of the most common cardiovascular diseases, and the incidence is increasing year by year. Many studies have shown that long non-coding RNA plays a vital role in the pathogenesis of AS. This study aimed to explore the role and mechanism of lncRNA-small nucleolar RNA host gene 8 (SNHG8) in AS. The expressions of serum lncSNHG8 and miR-224-3p were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic meaning of lncSNHG8 in AS was estimated by Receiver operating characteristic (ROC) curve. The correlation between lncSNHG8 and various clinical indicators, as well as miR-244-3p was evaluated by Pearson correlation coefficient analysis. Cell proliferation and migration were estimated by cell counting kit-8 (CCK-8) and Transwell assay. The interaction between lncSNHG8 and miR-224-3p was proved by luciferase reporter gene assay. The expression level of lncSNHG8 was increased in AS patients, while miR-224-3p expression was decreased. The ROC curve indicated that lncSNHG8 with high serum expression had the ability to distinguish AS. Pearson correlation coefficient exhibited that the level of miR-224-3p was negatively correlated with the level of lncSNHG8. The results of cell experiments indicated that inhibition of the expression of lncSNHG8 significantly inhibited the proliferation and migration of vascular smooth muscle cells (VSMCs). Luciferase reporter gene experiments confirmed that there was a target relationship between lncSNHG8 and miR-224-3p. In conclusion, lncSNHG8 had high diagnostic value for AS. It promoted the proliferation and migration of VSMCs by adsorption and inhibition of miR-224-3p.

Context: The effect of kaempferol, a regulator of oestrogen receptors, on atherosclerosis (AS) and the underlying mechanism is elusive., Objective: To explore the effect and mechanism of kaempferol on AS., Methods and Materials: In vivo , C57BL/6 and apolipoprotein E (APOE) -/- mice were randomly categorized into six groups (C57BL/6: control, ovariectomy (OVX), high-fat diet (HFD); APOE -/- : OVX-HFD, OVX-HFD + kaempferol (50 mg/kg) and OVX-HFD + kaempferol (100 mg/kg) and administered with kaempferol for 16 weeks, intragastrically. Oil-Red and haematoxylin-eosin (HE) staining were employed to examine the effect of kaempferol. In vitro , human aortic endothelial cells (HAECs) were pre-treated with or without kaempferol (5, 10 or 20 μM), followed by administration with kaempferol and oxidized low-density lipoprotein (ox-LDL) (200 μg/mL). The effect of kaempferol was evaluated using flow cytometry, and TdT-mediated dUTP Nick-End Labelling (TUNEL)., Results: In vivo , kaempferol (50 and 100 mg/kg) normalized the morphology of blood vessels and lipid levels and suppressed inflammation and apoptosis. It also activated the G protein-coupled oestrogen receptor (GPER) and PI3K/AKT/nuclear factor-erythroid 2-related factor 2 (Nrf2) pathways. In vitro , ox-LDL (200 μg/mL) reduced the cell viability to 50% (IC 50 ). Kaempferol (5, 10 or 20 μM) induced-GPER activation increased cell viability to nearly 10%, 19.8%, 30%, and the decreased cellular reactive oxygen species (ROS) generation (16.7%, 25.6%, 31.1%), respectively, consequently attenuating postmenopausal AS. However, the protective effects of kaempferol were blocked through co-treatment with si-GPER., Conclusions: The beneficial effects of kaempferol against postmenopausal AS are associated with the PI3K/AKT/Nrf2 pathways, mediated by the activation of GPER.

Vascular events can trigger a pathological phenotypic switch in vascular smooth muscle cells (VSMCs), decreasing and disrupting the plasticity and diversity of vascular networks. The development of novel therapeutic approaches is necessary to prevent these changes. We aimed to investigate the effects and associated mechanisms of low-intensity pulsed ultrasound (LIPUS) irradiation on the angiotensin II (AngII)-induced phenotypic switch in VSMCs. In vivo, AngII was infused subcutaneously for 4 weeks to stimulate vascular remodeling in mice, and LIPUS irradiation was applied for 20 min every 2 days for 4 weeks. In vitro, cultured rat aortic VSMCs (RAVSMCs) were pretreated once with LIPUS irradiation for 20 min before 48-h AngII stimulation. Our results showed that LIPUS irradiation prevents AngII-induced vascular remodeling of the whole wall artery without discriminating between adventitia and media in vivo and RAVSMC phenotypic switching in vitro. LIPUS irradiation downregulated miR-17-5p expression and upregulated peroxisome proliferator-activated receptor gamma (PPAR-γ) expression. The PPAR-γ activator rosiglitazone could mimic the favorable effects of LIPUS irradiation on AngII-treated RAVSMCs. In contrast, GW9662 could impede the LIPUS-mediated downregulation of RAVSMC proliferation and inflammation under AngII stimulation conditions in vivo and in vitro. Also, the miR-17-5p agomir has the same effects as GW9662 in vitro. Besides, the inhibitory effects of GW9662 against the anti-remodeling effects of LIPUS irradiation in AngII-induced RAVSMCs could be blocked by pretreatment with the miR-17-5p antagomir. Overall, LIPUS irradiation prevents AngII-induced RAVSMCs phenotypic switching through hampering miR-17-5p and enhancing PPAR-γ, suggesting a new approach for the treatment of vascular disorders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acute myocardial infarction (AMI) is a severe cardiovascular disease with high mortality. It is reported to be closely related to the mitochondrial dysfunction and metabolic disturbance on endothelial cells under a chronic hypoxic state. Significant declined mitochondrial respiration, ATP production, and metabolic changes are the main characteristics of endothelial injury in the disease. Trelagliptin is a DPP-4 inhibitor applied for the treatment of type II diabetes and has been recently reported to exert various pharmacological properties. In this investigation, we examined whether Trelagliptin possessed a protective effect against mitochondrial dysfunction and metabolic disturbance in human aortic valvular endothelial cells (HAVECs) under oxygen-glucose deprivation/reperfusion (OGD/R) conditions. We found that both the cytotoxicity and mitochondrial oxidative stress in HAVECs induced by OGD/R stimulation were greatly alleviated by Trelagliptin. In addition, the declined mitochondrial respiration and ATP production decreased secretion of cystathionine and creatine, and the increased production of triglyceride and adiponectin in OGD/R-challenged HAVECs was dramatically reversed by Trelagliptin, accompanied by the upregulated expression level of PGC-1α and CPT-1. Lastly, the AMPK pathway was observed to be significantly activated in OGD/R-challenged HAVECs by Trelagliptin treatment. After co-administration of the inhibitor of the AMPK pathway, the effects of Trelagliptin on mitochondrial function and metabolic alterations were significantly abolished. Taken together, our data indicate that Trelagliptin ameliorated OGD/R-induced mitochondrial disturbance and metabolic changes by activating the AMPK pathway., (© 2021. Japan Human Cell Society.)

Rapid endothelialization and regulation of smooth muscle cell proliferation are crucial for small-diameter vascular grafts to address poor compliance, thromboembolism, and intimal hyperplasia, and achieve revascularization. As a gaseous signaling molecule, nitric oxide (NO) regulates cardiovascular homeostasis, inhibits blood clotting and intimal hyperplasia, and promotes the growth of endothelial cells. Due to the instability and burst release of small molecular NO donors, a novel biomacromolecular donor has generated increasing interest. In the study, a low toxic NO donor of S-nitrosated keratin (KSNO) was first synthesized and then coelectrospun with poly(ε-caprolactone) to afford NO-releasing small-diameter vascular graft. PCL/KSNO graft was capable to generate NO under the catalysis of ascorbic acid (Asc), so the graft selectively elevated adhesion and growth of human umbilical vein endothelial cells (HUVECs), while inhibited the proliferation of human aortic smooth muscle cells (HASMCs) in the presence of Asc. In addition, the graft displayed significant antibacterial properties and good blood compatibility. Animal experiments showed that the biocomposite graft could inhibit thrombus formation and preserve normal blood flow via single rabbit carotid artery replacement for 1 month. More importantly, a complete endothelium was observed on the lumen surface. Taken together, PCL/KSNO small-diameter vascular graft has potential applications in vascular tissue engineering with rapid endothelialization and vascular remolding., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Endothelial dysfunction is the pathological basis of atherosclerosis. Incomplete understanding of endothelial dysfunction etiology has impeded drug development for this devastating disease despite the currently available therapies. Floralozone, an aroma flavor, specifically exists in rabbit ear grass. Recently, floralozone has been demonstrated to inhibit atherosclerosis, but the underlying mechanisms are undefined. The present study was undertaken to explore whether floralozone pharmacologically targets endothelial dysfunction and therefore exerts therapeutic effects on atherosclerosis. The Na+/H+ exchanger 1 (NHE1), a channel protein, plays a vital role in atherosclerosis. Whether NHE1 is involved in the therapeutic effects of floralozone on endothelial dysfunction has yet to be further answered. By performing oil red staining and hematoxylin-eosin staining, vascular functional study, and oxidative stress monitoring, we found that floralozone not only reduced the size of carotid atherosclerotic plaque but also prevented endothelial dysfunction in atherosclerotic rats. NHE1 expression was upregulated in the inner membrane of carotid arteries and H2O2-induced primary rat aortic endothelial cells. Inspiringly, floralozone prevented the upregulation of NHE1 in vivo and in vitro. Notably, the administration of NHE1 activator LiCl significantly weakened the protective effect of floralozone on endothelial dysfunction in vivo and in vitro. Our study demonstrated that floralozone exerted its protective effect on endothelial dysfunction in atherosclerosis by ameliorating NHE1. NHE1 maybe a drug target for the treatment of atherosclerosis, and floralozone may be an effective drug to meet the urgent needs of atherosclerosis patients by dampening NHE1., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The renin-angiotensin system, with the octapeptide angiotensin II as key player, is important in the renal, cardiac and vascular physiology. Prolyl carboxypeptidase (PRCP), prolyl endopeptidase (PREP) and angiotensin converting enzyme 2 (ACE2) are reported to be involved in the conversion of angiotensin II to angiotensin (1-7). Previous investigations showed that the processing of angiotensin II is cell- and species-specific and little is known about its conversion in human endothelial cells. Therefore, we aimed to investigate the C-terminal processing of angiotensin II and III in comparison to the processing of des-Arg 9 -bradykinin in human endothelial cells. To this end, human umbilical vein and aortic endothelial cells (HUVEC and HAoEC) were incubated with the peptides for different time periods. Mass spectrometry analysis was performed on the supernatants to check for cleavage products. Contribution of PRCP, ACE2 and PREP to the peptide cleavage was evaluated by use of the selective inhibitors compound 8o, DX600 and KYP-2047. The use of these selective inhibitors revealed that the C-terminal cleavage of angiotensin II and III was PRCP-dependent in HUVEC and HAoEC. In contrast, the C-terminal cleavage of des-Arg 9 -bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. With this study, we contribute to a better understanding of the processing of peptides involved in the alternative renin-angiotensin system. We conclude that PRCP is the main enzyme for the C-terminal processing of angiotensin peptides in human umbilical vein and aortic endothelial cells. For the first time the contribution of PRCP was investigated by use of a selective PRCP-inhibitor., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The arterial wall consists of three concentric layers: intima, media, and adventitia. Beyond their resident cells, these layers are characterized by an extracellular matrix (ECM), which provides both biochemical and mechanical support. Elastin, the major component of arterial ECM, is present in the medial layer and organized in concentric elastic lamellae that confer resilience to the wall. We explored the arterial wall structures from C57Bl6 (control), db/db (diabetic), and ApoE -/- (atherogenic) mice aged 3 months using synchrotron X-ray computed microtomography on fixed and unstained tissues with a large image field (8 mm 3 ). This approach combined a good resolution (0.83 µm/voxel), large 3D imaging field. and an excellent signal to noise ratio conferred by phase-contrast imaging. We determined from 2D virtual slices that the thickness of intramural ECM structures was comparable between strains but automated image analysis of the 3D arterial volumes revealed a lattice-like network within concentric elastic lamellae. We hypothesize that this network could play a role in arterial mechanics. This work demonstrates that phase-contrast synchrotron X-ray computed microtomography is a powerful technique which to characterize unstained soft tissues., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Aging and smoking are major risk factors for cardiovascular diseases (CVD). Our in vitro study compared, in the context of aging, the effects of the aerosol of Tobacco Heating System 2.2 (THS; an electrically heated tobacco product) and 3R4F reference cigarette smoke (CS) on processes that contribute to vascular pathomechanisms leading to CVD. Young and old human aortic smooth muscle cells (HAoSMC) were exposed to various concentrations of aqueous extracts (AE) from 3R4F CS [0.014-0.22 puffs/mL] or THS aerosol [0.11-1.76 puffs/mL] for 24 h. Key markers were measured by high-content imaging, transcriptomics profiling and multianalyte profiling. In our study, in vitro aging increased senescence, DNA damage, and inflammation and decreased proliferation in the HAoSMCs. At higher concentrations of 3R4F AE, young HAoSMCs behaved similarly to aged cells, while old HAoSMCs showed additional DNA damage and apoptosis effects. At 3R4F AE concentrations with the maximum effect, the THS AE showed no significant effect in young or old HAoSMCs. It required an approximately ten-fold higher concentration of THS AE to induce effects similar to those observed with 3R4F. These effects were independent of nicotine, which did not show a significant effect on HAoSMCs at any tested concentration. Our results show that 3R4F AE accelerates aging in young HAoSMCs and exacerbates the aging effect in old HAoSMCs in vitro, consistent with CS-related contributions to the risk of CVD. Relative to 3R4F AE, the THS AE showed a significantly reduced impact on HAoSMCs, suggesting its lower risk for vascular SMC-associated pathomechanisms leading to CVD., (© 2021. The Author(s).)

Telocytes (TCs), a novel interstitial cell entity promoting tissue regeneration, have been described in various tissues. Their role in inter-cellular signalling and tissue remodelling has been reported in almost all human tissues. This study hypothesizes that TC also contributes to tissue remodelling and regeneration of the human thoracic aorta (HTA). The understanding of tissue homeostasis and regenerative potential of the HTA is of high clinical interest as it plays a crucial role in pathogenesis from aortic dilatation to lethal dissection. Therefore, we obtained twenty-five aortic specimens of heart donors during transplantation. The presence of TCs was detected in different layers of aortic tissue and characterized by immunofluorescence and transmission electron microscopy. Further, we cultivated and isolated TCs in highly differentiated form identified by positive staining for CD34 and c-kit. Aortic-derived TC was characterized by the expression of PDGFR-α, PDGFR-β, CD29/integrin β-1 and αSMA and the stem cell markers Nanog and KLF-4. Moreover, TC exosomes were isolated and characterized for soluble angiogenic factors by Western blot. CD34 + /c-kit + TCs shed exosomes containing the soluble factors VEGF-A, KLF-4 and PDGF-A. In summary, TC occurs in the aortic wall. Correspondingly, exosomes, derived from aortic TCs, contain vasculogenesis-relevant proteins. Understanding the regulation of TC-mediated aortic remodelling may be a crucial step towards designing strategies to promote aortic repair and prevent adverse remodelling., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)