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Haemophilus parasuis can elicit serious inflammatory responses, which contribute to huge economic losses to the swine industry. However, the pathogenic mechanisms underlying inflammation-related damage induced by H. parasuis remain unclear. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) have important functions in the regulation of autoimmune disorders. Baicalin has been shown to have anti-inflammatory, anti-microbial, and anti-oxidant activities. In this study, we investigated whether lncRNAs were involved in the vascular injury or inflammation triggered by H. parasuis and whether baicalin regulated the lncRNA profiles of porcine aortic vascular endothelial cells (PAVECs) infected with H. parasuis . The results showed that the lncRNA and mRNA expression profiles of PAVECs were changed by H. parasuis . Important functions of lncRNAs and mRNAs were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that the targets of differentially expressed lncRNAs of H . parasuis infected PAVECs were mainly involved in the tumor necrosis factor (TNF) signaling pathway, apoptosis, and N-glycan biosynthesis; whereas nicotinate and nicotinamide metabolism, the cytosolic DNA-sensing pathway, the TNF signaling pathway, and the nuclear factor (NF)-kappa B signaling pathway were enriched in PAVECs pretreated with baicalin. In addition, top hub genes and lncRNAs were identified and validated by quantitative polymerase chain reaction. CCL5 , GBP1 , and SAMHD1 were significantly upregulated after H. parasuis infection, whereas they were significantly downregulated with baicalin pretreatment. LncRNA ALDBSSCT0000001677, ALDBSSCT0000001353, MSTRG.10724.2, and ALDBSSCT0000010434 had the same expression pattern. Collectively, these data suggested that baicalin could modify changes to the lncRNAs profiles or regulate lncRNAs that participate in inflammation-related signaling pathways, thereby alleviating tissue damage or inflammatory responses induced by H. parasuis . To our best knowledge, this is the first article of H. parasuis stimulating changes to the lncRNA profiles of PAVECs and the capability of baicalin to regulate lncRNA changes in PAVECs infected with H. parasuis , which might provide a novel therapeutic target for the control of H. parasuis infection.

Glucagon-like peptide 1 receptor (GLP-1R) belongs to the family B of G protein-coupled receptors (GPCRs) and has antidiabetic and cardioprotective effects. Classical GLP-1R at the plasma membrane undergoes desensitization and internalization and is recycled back to the plasma membrane under the control of GLP-1 in islet β-cells. However, the subcellular localization of GLP-1R in the vascular system remains unclear. Here, we find that GLP-1R is localized in the nucleus of rat aortic smooth muscle cells (RASMCs) and in the tunica media. We identify a functional nuclear localization signal (NLS; 412-442aa) at the C-terminal region of GLP-1R. Nuclear import of GLP-1R is mediated by an importin-α-dependent pathway and regulated by phosphorylation of Ser416 in the NLS. Upon leaving the nucleus, GLP-1R promotes cell proliferation in RASMCs. These findings may provide insights into the cardiovascular functions of GLP-1R., (© 2020 Federation of European Biochemical Societies.)

Vascular smooth muscle cells (VSMCs) proliferation and migration play a fundamental role during the process of hypertensive angiopathy. Angiotensin-II (Ang-II) is one of the robust phenotype-modulating agents, which changes VSMCs to efficiently proliferate and migrate. The mechanism of the proliferation and migration is not well understood yet. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. We approved that Septin4 expression was upregulated in mouse aorta by continuous infusion of Ang-II and in cultured VSMCs treated with Ang-II. Overexpression of Septin4 led to lower level of autophagy and decreased capacity of proliferation and migration. In order to identify the mechanism by which Septin4 interacts with these processes, we blocked autophagy by chloroquine (CQ). After inhibiting the autophagy, the ability of proliferation and migration was further restrained in the Septin4 overexpression VSMCs. In conclusion, our results indicated that during the process of VSMCs proliferation and migration induced by Ang-II, Septin4 modulated autophagy and thus regulated the activity of proliferation and migration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aims of this study were to assess whether ischemic preconditioning (PC) induces bradykinin (Bk) synthesis in bovine aortic endothelial cells (bAECs) and, if so, to explore the molecular mechanisms by which this peptide provides cytoprotection against hypoxia. PC was induced by exposing bAECs to three cycles of 15 min of hypoxia followed by 15 min of reoxygenation. Bk synthesis peaked in correspondence to the early and late phases of PC (10 -12 M and 10 -11 M, respectively) and was abolished by a selective tissue kallikrein inhibitor, aprotinin. Stimulation with exogenous Bk at concentrations of 10 -12 M and 10 -11 M reduced the cell death induced by 12 h of hypoxia by 50%. Pretreatment with HOE-140, a Bk receptor 2 (BKR2) inhibitor, in bAECs exposed to 12 h of hypoxia, abrogated the cytoprotective effect of early and late PC, whereas des-Arg-HOE-140, a Bk receptor 1 (BKR1) inhibitor, affected only the late PC. In addition, we found that PC evoked endocytosis and the recycling of BKR2 during both the early and late phases, and that inhibition of these pathways affected PC-mediated cytoprotection. Finally, we evaluated the activation of PKA and Akt in the presence or absence of BKR2 inhibitor. HOE-140 abrogated PKA and Akt activation during both early and late PC. Consistently, BKR2 inhibition abolished cross-talk between PKA and Akt in PC. In bAECs, Bk-synthesis evoked by PC mediates the protection against both apoptotic and necrotic hypoxia-induced cell death in an autocrine manner, by both BKR2- and BKR1-dependent mechanisms.

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is widely used to treat hypertension. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. A growing body of evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of telmisartan on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which telmisartan inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Telmisartan inhibited phenylephrine-induced vessel contraction in endothelium-deprived aortas, and decreased myosin light chain kinase (MLCK) levels (without altering corresponding mRNA levels) and myosin light chain (MLC) phosphorylation at Ser19 (p-MLC-Ser 19 ) in VSMCs. MG-132 but not doxycycline significantly restored telmisartan-inhibited MLCK expression and p-MLC-Ser 19 . Telmisartan induced AMP-activated protein kinase (AMPK) phosphorylation at Thr172 (p-AMPK-Thr 172 ), and compound C or ectopic expression of the dominant negative (dn)-AMPKα1 gene significantly reversed telmisartan-inhibited MLCK expression and p-MLC-Ser 19 . Of the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr 172 , and inhibited MLCK expression and p-MLC-Ser 19 . GW9662 had no effects on telmisartan-induced changes. Similar to the in vitro results, telmisartan enhanced p-AMPK-Thr 172 , and inhibited MLCK expression and p-MLC-Ser 19 in endothelium-deprived aortas. Furthermore, the telmisartan-inhibited vessel contraction in the aortas was significantly reversed by MG-132 or compound C. In conclusion, we demonstrated that telmisartan inhibits VSMC contractility and vessel contraction by activating AMPK/proteasome/MLCK degradation signaling axis. These results suggest that telmisartan can be used to treat pathological vasospasms., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: To investigate the effect of androgens and estrogens on surtuin 1 (SIRT1) expression in human aortic endothelial cells (HAECs)., Methods: Real-time polymerase chain reaction analysis of SIRT-1 expression over 48 hours (h) was performed in HAECs treated with various concentrations of dehydroepiandrostendione (DHEA), androstenedione and testosterone (androgens), estrone (E1), estradiol (E2), and estriol (E3) (estrogens) to investigate the dose-dependency of time courses. The influence of high glucose on SIRT1 expression induced by the androgens and estrogens was also examined., Results: Dehydroepiandrostendione, androstenedione, and testosterone remarkedly produced a dose-dependent increase in SIRT1 expression in the range of 10 to 20 μg/ml. High glucose (40mM) medium had significantly inhibitory effects on 10 μg/ml DHEA-induced SIRT1 expression (p=0.024). Estrone and E2, but not E3, caused a marked dose-dependent increase in SIRT1 expression from 10 to 20 μg/ml. Treatment with 20 mM or 40 mM glucose medium did not significantly inhibit E1- and E3-induced SIRT1 expression in control medium; however, both high glucose mediums significantly emphasized E2-induced SIRT1 expression in control medium (p=0.007, p=0.005)., Conclusion: These results suggest that DHEA, androstenedione, testosterone, E1, and E2 definitely activate SIRT1 expression in HAECs. A high glucose medium is potent to inhibit the basal gene expression; however, it could not reduce powerful androgen- and estrogen-induced SIRT1 expression in HAECs.

Vascular oxidative stress, inflammatory response, and proliferation are crucial mediators of vascular dysfunction which contribute to the pathology of hypertension. A tripeptide, LRW (Leu-Arg-Trp), was characterized from pea protein legumin, and its previously studied isomer IRW (Ile-Arg-Trp) was reported to exhibit antihypertensive activity via activation of angiotensin-converting enzyme 2. The objective of the current study was to explore the effects of LRW on vascular stress in vascular smooth muscle cells (VSMCs) under angiotensin II (Ang II)-induced cellular stress. LRW treatment could decrease Ang II-triggered superoxide production, inflammation, and proliferation in VSMCs. The abovementioned advantageous effects appeared to involve the upregulation of the ACE2-Ang-(1-7)-MasR axis and modulation of the nuclear factor-κB pathway. These findings specified the prospective role of LRW as a functional food ingredient or nutraceutical in the prevention of cardiovascular diseases, particularly hypertension and vascular damage.

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD + /NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO - group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD + -dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2-AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Caon et al.)

Small extracellular vesicles (sEV) contain various molecules and mediate cell-to-cell communication under both physiological and pathological conditions. We have recently reported that sEV isolated from plasma of normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) regulate systemic blood pressure. The initiation and development of hypertension partly rely on proliferation and migration of vascular smooth muscle cells (SMCs) followed by the structural remodeling of vascular wall. In the present study, we examined the effects of plasma sEV in WKY and SHR on the proliferative and migratory functions of primary rat aortic SMCs. There was no difference in the concentration and size distribution of plasma sEV between WKY and SHR, while the protein expression of CD81 in plasma sEV from SHR was lower than that from WKY. Both plasma sEV from WKY and SHR were internalized into SMCs and stimulated the migration and proliferation with a similar potency. In summary, we, for the first time, demonstrated that plasma sEV in WKY and SHR are physiologically active in terms of proliferative and migratory functions, however, these effects do not seem to be related to the pathogenesis of hypertension development.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm. In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre +/- ;Rosa26 RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP + neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3-5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

The unfolded protein response element protects against endoplasmic reticulum stress and delivers protection towards potentially harmful challenges. The components of this multi-branch molecular machinery, namely the protein kinase RNA-like ER kinase, the activating transcription factor 6, and the inositol-requiring enzyme-1α; expand the endoplasmic reticulum capacity to support cellular function under stress conditions. In the present study, we employed bovine pulmonary aortic endothelial cells and mice to investigate the possibility that the Hsp90 inhibitors Tanespimycin (17-AAG) and Luminespib (AUY-922) exert the capacity to trigger the unfolded protein response. The induction of the unfolded protein response regulators immunoglobulin heavy-chain-binding protein, endoplasmic reticulum oxidoreductin-1alpha; and protein disulfide isomerase was also examined. It appears that both inhibitors capacitate the induction of the unfolded protein response element in vitro, since lung cells exposed to 1, 2 and 10 μM of 17-AAG or AUY-922 for 4, 6, 8, 16 and 48 h demonstrated increased levels of those proteins. Similar events occurred in the lungs of mice treated with AUY-922. Thus, our study demonstrates that Hsp90 inhibition triggers the activities of the unfolded protein response, and suggests that this molecular machinery contributes in the protective action of Hsp90 inhibitors in the lung microvasculature., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Improving diagnostic imaging and therapy by targeted compound delivery to pathological areas and across biological barriers is of urgent need. A lipopeptide, P-CrA-A2, composed of a highly cationic peptide sequence (A2), an N-terminally attached palmitoyl chain (P) and cryptophane molecule (CrA) for preferred uptake into blood-brain barrier (BBB) capillary endothelial cells, was generated. CrA allows reversible binding of Xe for NMR detection with hyperpolarized nuclei. The lipopeptide forms size-optimized micelles with a diameter of about 11 nm at low micromolar concentration. Their high local CrA payload has a strong and switchable impact on the bulk magnetization through Hyper-CEST detection. Covalent fixation of CrA does not impede micelle formation and does not hamper its host functionality but simplifies Xe access to hosts for inducing saturation transfer. Xe Hyper-CEST magnetic resonance imaging (MRI) allows for distinguishing BBB endothelial cells from control aortic endothelial cells, and the small micelle volume with a sevenfold improved CrA-loading density compared to liposomal carriers allows preferred cell labelling with a minimally invasive volume (≈16 000-fold more efficient than 19 F cell labelling). Thus, these nanoscopic particles combine selectivity for human brain capillary endothelial cells with great sensitivity of Xe Hyper-CEST MRI and might be a potential MRI tool in brain diagnostics., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lysophosphatidylcholine (lysoPtdCho) is produced by the phospholipase A 2 -mediated hydrolysis of phosphatidylcholine and can stimulate proliferation and apoptosis of vascular smooth muscle cells. We examined the influence of fetal bovine serum (FBS) concentration in the culture medium on lysoPtdCho-mediated apoptosis and proliferation of human aortic smooth muscle cells (HASMCs) as well as on the activation of extracellular signal-regulated kinases (ERK)1/2. In the presence of 1% FBS, HASMC viability increased after lysoPtdCho treatment at 1 and 10 μM but decreased at 25 and 50 μM. However, lysoPtdCho increased HASMC viability in a dose-dependent manner in the presence of 10% FBS. The activity of caspase 3/7 in HASMCs was increased by 25 μM lysoPtdCho in the presence of 1% FBS, but not 10% FBS. Furthermore, lysoPtdCho at 1 and 10 μM triggered ERK1/2 phosphorylation in the presence of 1% FBS, but not at 10% FBS. Thus, lysoPtdCho-mediated HASMC apoptosis, proliferation, and ERK1/2 activation are dependent on the concentration of FBS.

β-Sitosterol is a natural compound widely found in many vegetable oils, nuts, and plant medicines; it lowers the cholesterol levels, enhances the production of plasminogen activators, and exhibits anticancer and antiatherogenic effects. However, the direct endothelial protection of β-sitosterol against an oxidized low-density lipoprotein (ox-LDL) is not well understood. In the present study, β-sitosterol significantly inhibited cell apoptosis (P < 0.01), increased cell migration (P < 0.01), improved energy metabolism (P < 0.05) and improved morphology after ox-LDL (50 μg ml-1) exposure following β-sitosterol (2 μg mL-1) treatment in human aortic endothelial cells (HAECs ). A total of 691 differentially expressed (DE) mRNAs were identified (579 were upregulated and 112 were downregulated, fold change ≥2.0, P < 0.05) after 24 h of β-sitosterol administration in transcriptome sequencing (β-sitosterol vs. ox-LDL), which suggested that β-sitosterol reversed 62.32% change in mRNAs induced by ox-LDL. DE mRNAs are enriched mainly in focal adhesion, ribosomes, eukaryotic translation elongation, etc. Considering that one of the enrichment is 3'-UTR-mediated translational regulation, we explored DE microRNA (miRNA). The miRNA-seq data proposed 87 up-regulated and 58 down-regulated miRNAs (fold change ≥2.0, P < 0.05) in miRNA-seq (β-sitosterol vs. ox-LDL), suggesting that β-sitosterol reversed 76.67% change in miRNAs induced by ox-LDL. The DE miRNA-DE mRNA coexpression network focused on ribosomes, cell cycle, oxidative phosphorylation, PI3K-Akt signaling pathway, TNF signaling pathway, ErbB signaling pathway, and mTOR signaling pathway. Consequently, miRNAs might be the targets of β-sitosterol and play vital roles in transcriptional regulation in endothelial protective and antiatherogenic effects against ox-LDL.

In-stent restenosis is a serious concern for patients treated through the stenting procedure, although this can be solved using drug-eluting stents and/or drug-eluting balloon catheters. However, the chemical agents released from the drug-eluting layer for inhibiting smooth muscle cell (SMC) migration are inevitably associated with damage to vascular endothelial cell (ECs). The present in vitro study used a distinct strategy, in which a smart gene (phEGR1-PKCδ, an engineered plasmid consists of an SMC-specific promoter (human early growth response 1, hEGR1 promoter) ligated with a gene encoding apoptosis-inducing protein (protein kinase C-delta, PKCδ) was incorporated into a novel gene vehicle (Au cluster-incorporated polyethylenimine/carboxymethyl hexanoyl chitosan, PEI-Au/CHC) to form the PEI-Au/CHC/phEGR1-PKCδ complex, which was proposed for the selective inhibition of SMC proliferation. It was found that the cell viability of SMCs receiving the PEI-Au/CHC/phEGR1-PKCδ complex under simulated inflammation conditions was significantly lower than that of the ECs receiving the same treatment. In addition, the PEI-Au/CHC/phEGR1-PKCδ complex did not demonstrate an inhibitory effect on EC proliferation and migration under simulated inflammation conditions. Finally, the PEI-Au/CHC/phEGR1-PKCδ complexes coated onto a balloon catheter used in percutaneous transluminal coronary angioplasty (PTCA) could be transferred to both the ECs and the SMC layer of Sprague Dawley (SD) rat aortas ex vivo. These preliminary in vitro results suggest that the newly developed approach proposed in the present study might be a potential treatment for reducing the incidence rate of in-stent restenosis and late thrombosis in the future.

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41 + HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production., Competing Interests: Declaration of Interests V.L. is currently at the Karolinska Institutet, L.N.T. is at Sanofi, and A.G.C. is at the Peter MacCallum Cancer Centre. G.Q.D. holds equity interest in True North Therapeutics and 28/7 Therapeutics; D.E.I. holds equity in Emulate Inc. and chairs its scientific advisory board., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bone marrow-derived stem/progenitor cells have been utilized for cardiac or vascular repair after ischemic injury, but they are subject to apoptosis and immune rejection in the ischemic site. Multiple scaffolds were used as delivery tools to transplant stem/progenitor cells; however, these scaffolds did not show intrinsically antiapoptotic or anti-inflammatory properties. Decellularized aortic scaffolds that facilitate cell delivery and tissue repair were prepared by removing cells of patient-derived aortic tissues. Scanning electron microscopy (SEM) showed cells attached well to the scaffold after culturing for 5 days. Live/dead staining showed most seeded cells survived at day 7 on a decellularized aortic scaffold. Ki67 staining demonstrated that decellularized aortic scaffold promoted proliferation of bone marrow-derived CD34+ progenitor cells. Apoptosis of CD34+ progenitor cells induced by H 2 O 2 at high concentration was significantly alleviated in the presence of decellularized aortic scaffolds, demonstrating a protective effect against oxidative stress-induced apoptosis. Furthermore, decellularized aortic scaffolds significantly reduced the expression of proinflammatory cytokines (IL-8, GM-CSF, MIP-1 β , GRO- α , Entoxin, and GRO) concurrently with an increase in anti-inflammatory cytokines (IL-2 and TGF- β ) released from CD34+ progenitor cells when exposed to H 2 O 2 at low concentration. Finally, neovascularization was observed by H&E and immunohistochemical staining 14 days after the decellularized aortic scaffolds were subcutaneously implanted in nude mice. This preclinical study demonstrates that the use of a decellularized aortic scaffold possessing antiapoptotic and anti-inflammatory properties may represent a promising strategy for cardiovascular repair after ischemic injury., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Liping Gao et al.)

Although EHMT2 (also known as G9a) plays a critical role in several kinds of cancers and cardiac remodeling, its function in vascular smooth muscle cells (VSMCs) remains unknown. In the present study, we revealed a novel function of EHMT2 in regulating autophagic cell death (ACD) of VSMC. Inhibition of EHMT2 by BIX01294 or knockdown of EHMT2 resulted in reduced VSMC numbers which were independent of proliferation and apoptosis. Interestingly, EHMT2 protein levels were significantly decreased in VSMCs treated with autophagic inducers. Moreover, more autophagic vacuoles and accumulated LC3II were detected in VSMCs treated with BIX01294 or lenti-shEHMT2 than their counterparts. Furthermore, we found that EHMT2 inhibited the ACD of VSMCs by suppressing autophagosome formation. Mechanistically, the pro-autophagic effect elicited by EHMT2 inhibition was associated with SQSTM1 and BECN1 overexpression. Moreover, these detrimental effects were largely nullified by SQSTM1 or BECN1 knockdown. More importantly, similar results were observed in primary human aortic VSMCs. Overall, these findings suggest that EHMT2 functions as a crucial negative regulator of ACD via decreasing SQSTM1 or BECN1 expression and that EHMT2 could be a potent therapeutic target for cardiovascular diseases ( e.g., aortic dissection)., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background : The LRP1 (CR9) domain and, in particular, the sequence Gly 1127 -Cys 1140 (P3) plays a critical role in the binding and internalization of aggregated LDL (agLDL). We aimed to evaluate whether immunization with P3 reduces high-fat diet (HFD)-induced atherosclerosis. Methods : Female New Zealand White (NZW) rabbits were immunized with a primary injection and four reminder doses (R1-R4) of IrP (irrelevant peptide) or P3 conjugated to the carrier. IrP and P3-immunized rabbits were randomly divided into a normal diet group and a HFD-fed group. Anti-P3 antibody levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular pro-inflammatory mediators were determined using standardized methods while atherosclerosis was determined by confocal microscopy studies and non-invasive imaging (PET/CT and Doppler ultrasonography). Studies treating human macrophages (hMΦ) and coronary vascular smooth muscle cells (hcVSMC) with rabbit serums were performed to ascertain the potential impact of anti-P3 Abs on the functionality of these crucial cells. Results : P3 immunization specifically induced the production of anti-P3 antibodies (Abs) and did not alter the lipoprotein profile. HFD strongly induced cholesteryl ester (CE) accumulation in the aorta of both the control and IrP groups, and their serum dose-dependently raised the intracellular CE of hMΦ and hcVSMC, promoting TNFR1 and phospho-NF-kB (p65) overexpression. These HFD pro-inflammatory effects were dramatically decreased in the aorta of P3-immunized rabbits and in hMΦ and hcVSMC exposed to the P3 rabbit serums. Microscopy studies revealed that P3 immunization reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and lesion area in the aorta. PET/CT and Doppler ultrasonography studies showed that the average standardized uptake value (SUV mean ) of the aorta and the arterial resistance index (ARI) of the carotids were more upregulated by HFD in the control and IrP groups than the P3 group. Conclusions : P3 immunization counteracts HFD-induced fatty streak formation in rabbits. The specific blockade of the LRP1 (CR9) domain with Anti-P3 Abs dramatically reduces HFD-induced intracellular CE loading and harmful coupling to pro-inflammatory signaling in the vasculature., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pathological studies have shown that the vulnerability of plaques affects outcomes in patients with atherosclerosis (AS), a chronic inflammatory disease and common cause of morbidity and mortality worldwide. Although emerging technologies have enabled early diagnosis of AS with high-risk vulnerable plaques, more accurate and noninvasive diagnostic methods are urgently required. To this end, molecules involved in genetic or epigenetic regulation of the vulnerability of atherosclerotic plaques have been extensively studied. Here, we evaluated long noncoding RNA (lncRNA) variability by microarray assay in murine aortic endothelial cells (MAECs) bearing vulnerable plaques and identified the novel functional lncRNA UC.98, whose expression pattern was associated with the vulnerability of atherosclerotic plaques. Consistent with this, clinical statistics comparing the peripheral blood specimens from sets of patients with AS with or without vulnerable plaques confirmed the linear relationship between the expression pattern of UC.98 and plaque instability. Moreover, MTT assays and western blot analysis showed that silencing of intrinsic UC.98 in MAECs not only suppressed cell proliferation but also decreased the expressions of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, thereby inactivating the nuclear factor-κB pathway. In conclusion, our results highlighted the pivotal role of UC.98 in regulating the vulnerability of plaques during AS progression and suggested that UC.98 may be a biomarker of the early diagnosis and prognosis of AS with vulnerable plaques and a potential therapeutic target for slowing AS progression., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study investigated the effect of acylated ghrelin (AG) deficiency after sleeve gastrectomy (SG) or chronic administration in control and SG-indiuced rats on platelet function, coagulation, and fibrinolysis. Administration of AG (100 µg/kg, subcutaneously) to control or SG rats significantly inhibited platelets aggregation and lowered levels of Von-Willebrand factor (vWF), fibrinogen, and thromboxane B2. Concomitantly, it decreased circulatory levels and aortic expression levels of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) and increased the aortic expression of the endothelial nitric oxidase (eNOS). However, AG inhibited angiotensin-II (ANGII)-induced upregulation of tissue factor pathway inhibitor (TPAI) and TF and increased activity of TF and increases eNOS expression in cultured endothelial cells, an effect that was abolished by the addition of D-[lys3]-GHRP-6, a selective AG receptor (GHSR-1a) blocker or L-Name, a potent eNOS inhibitor. In conclusion, AG has an anti-platelet, anti-coagulant, and fibrinolytic roles mediated through GHSR-1a to enhance nitric oxide synthesis.

Type 2 diabetes is associated with oxidative stress and low-grade inflammation resulting in endothelial dysfunction (ED). This study determined to explore the protective effects of berry-derived anthocyanins (AC) with potent antioxidant and anti-inflammatory activities in human diabetic endothelial cells upon oxidative and inflammatory stressors. Cultured healthy human aortic endothelial cells (HAEC) and diabetic human aortic endothelial cells (D-HAEC) exposed to oxidative stress by hydrogen peroxide (H 2 O 2 , 75 μM) and lipopolysaccharide (LPS, 1 μg/mL) as an inflammatory inducer before treatment with AC (50 μl/ml). The results from cytotoxicity assays showed that AC had no significant effects in cell viability (P-value < 0.0001), and exposure to H 2 O 2 75 μM had a less toxic effect (P-value < 0.05). Although, AC significantly decreased H 2 O 2 -induced cytotoxicity and oxidative stress in both HAEC and D-HAEC cell lines (P-value < 0.0001), no positive impact of AC was found on the GSSG/GSH ratios (P-value < 0.05). Exposure to the LPS increased the production of IL-6 in both HAEC and D-HAEC cell lines (P-value < 0.0001), whereas AC treatment reduced LPS-induced IL-6 production in both cell lines with a more robust impact on D-HAEC (P-value < 0.0001). While LPS increased inflammasome assembling and caspase-1 activation, AC treatment inhibited caspase-1 activation in D-HAEC (P ≤ 0.05). This study indicated that berry anthocyanins reduced oxidative stress and inflammation via the inhibition of the NF-ƙB signaling pathway, which contributes to mitigating the diabetes-induced up-regulation of NF-ƙB., 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 © 2020 Elsevier B.V. All rights reserved.)

Human diving is known to induce endothelial dysfunction. The aim of this study was to decipher the mechanism of ROS production during diving through the measure of mitochondrial calcium concentration, peroxynitrite, NO°, and superoxide towards better understanding of dive-induced endothelial dysfunction. Air diving simulation using bovine arterial endothelial cells (compression rate 101 kPa/min to 808 kPa, time at depth 45 min) was performed in a system allowing real-time fluorescent measurement. During compression, the cells showed increased mitochondrial superoxide, peroxynitrite, and mitochondrial calcium, and decreased NO° concentration. MnTBAP (peroxynitrite scavenger) suppressed superoxide, recovered NO° production and promoted stronger calcium influx. Superoxide and peroxynitrite were inhibited by L-NIO (eNOS inhibitor), but were further increased by spermine-NONOate (NO° donor). L-NIO induced stronger calcium influx than spermine-NONOate or simple diving. The superoxide and peroxynitrite were also inhibited by ruthenium red (blocker of mitochondrial Ca 2+ uniporter), but were increased by CGP (an inhibitor of mitochondrial Na + -Ca 2+ exchange). Reactive oxygen and nitrogen species changes are associated, together with calcium mitochondrial storage, with endothelial cell dysfunction during simulated diving. Peroxynitrite is involved in NO° loss, possibly through the attenuation of eNOS and by increasing superoxide which combines with NO° and forms more peroxynitrite. In the field of diving physiology, this study is the first to unveil a part of the cellular mechanisms of ROS production during diving and confirms that diving-induced loss of NO° is linked to superoxide and peroxynitrite.

Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. Linc-ROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4-6%, 2-8% and 0-10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Angiopoietin-1 (Ang-1) is an important proangiogenic factor also involved in the maintenance of endothelial-barrier integrity. The small GTPase Rap1 is involved in the regulation of adherens junctions through VE-cadherin-mediated adhesion, and in endothelial permeability. While many studies established that Rap1 activation is critical for endothelial cell-cell adhesions, its roles in the antipermeability effects of Ang-1 are ill-defined. Thus, we determined the contribution of Rap1 to Ang-1-stimulated angiogenic effects on endothelial cells (ECs). We found that Rap1 is activated following Ang-1 stimulation and is required for the antipermeability effects of Ang-1 on EC monolayers. Our results also revealed that Rap1 is necessary for EC sprouting stimulated by Ang-1 but had no significant effect on Ang-1-induced EC migration and adhesion. In contrast, downregulation of VE-cadherin markedly increased the adhesiveness of ECs to the substratum, which resulted in inhibition of Ang-1-stimulated migration. These results revealed that Rap1 is central to the effects of Ang-1 at intercellular junctions of ECs, whereas VE-cadherin is also involved in the adhesion of ECs to the extracellular matrix., Competing Interests: The authors declare no conflict of interest.

NPCdc is a synthetic natriuretic peptide that was originally derived from another peptide, the NP2&#95;Casca, isolated from Crotalus durissus cascavella venom. These molecules share 70% structural homology with natriuretic peptides obtained from different species, including humans. NP2&#95;Casca induces vasorelaxation and increases nitric oxide levels independently of natriuretic peptide receptors A and B. This study aimed to investigate whether NPCdc-induced hypotension in control rats and rats with a reduced kidney mass is associated with effects on the glomerular filtration rate, NADPH oxidase activity and components downstream of natriuretic peptide receptor C (NPR-C). Anaesthetized Wistar rats that were subjected to a sham operation and 5/6 nephrectomy (5/6Nx) were infused with saline (vehicle) or NPCdc (7.5 μg/kg/min) for 70 min. The NPCdc treatment decreased the mean arterial pressure and NADPH oxidase activity while simultaneously increasing the glomerular filtration rate, fractional Na + excretion and nitric oxide level. After 70 min, the levels of p-AKT Ser-473 , p-eNOS Ser-1177 , p-nNOS Ser-1417 and p-iNOS Tyr-151 were not affected. However, p-ERK1/2 Thr-202/Tyr-204 levels were altered. Thus, nitric oxide and components of NPR-C signalling mediate the effects of NPCdc. The results suggest a potential therapeutic application of this peptide for cardiorenal syndrome., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The process of aortic injury, repair, and remodeling during aortic aneurysm and dissection is poorly understood. We examined the activation of bone marrow (BM)-derived and resident aortic cells in response to aortic injury in a mouse model of sporadic aortic aneurysm and dissection., Materials and Methods: Wild-type C57BL/6 mice were transplanted with green fluorescent protein (GFP)+ BM cells. For 4 wk, these mice were either unchallenged with chow diet and saline infusion or challenged with high-fat diet and angiotensin II infusion. We then examined the aortic recruitment of GFP+ BM-derived cells, growth factor production, and the differentiation potential of GFP+ BM-derived and GFP- resident aortic cells., Results: Aortic challenge induced recruitment of GFP+ BM cells and activation of GFP- resident aortic cells, both of which produced growth factors. Although BM cells and resident aortic cells equally contributed to the fibroblast populations, we did not detect the differentiation of BM cells into smooth muscle cells. Interestingly, aortic macrophages were both of BM-derived (45%) and of non-BM-derived (55%) origin. We also observed a significant increase in stem cell antigen-1 (Sca-1)+ stem/progenitor cells and neural/glial antigen 2 (NG2+) cells in the aortic wall of challenged mice. Although some of the Sca-1+ cells and NG2+ cells were BM derived, most of these cells were resident aortic cells. Sca-1+ cells produced growth factors and differentiated into fibroblasts and NG2+ cells., Conclusions: BM-derived and resident aortic cells are activated in response to aortic injury and contribute to aortic inflammation, repair, and remodeling by producing growth factors and differentiating into fibroblasts and inflammatory cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The interaction between nanohydroxyapatite (HAP) and smooth muscle cells is an important step in vascular calcification. However, the effect of the shape of HAP on adhesion and endocytosis to aortic smooth muscle cells has been rarely reported. Four different morphological HAP crystals (H-Rod, H-Needle, H-Sphere, and H-Plate) were selected to interact with rat aortic smooth muscle cells (A7R5). Fluorescence-labeled HAP was used to detect crystal adhesion and endocytosis and then pretreated with different endocytic inhibitors to explore the pathway of endocytotic crystals. The distribution of crystals inside and outside the cells and the crystal localization in lysosomes was observed through laser confocal microscopy. The effect of crystal on the cell cycle and the changes in the expression of phosphatidylserine, osteopontin, α-actin, core binding factor alpha 1, and osterix on the surface of A7R5 cells were detected. The adhesion and endocytosis of HAP on A7R5 cells were closely related to crystal shapes and ranked as follows: H-Plate > H-Sphere > H-Needle > H-Rod. H-Sphere and H-Needle were internalized into the cells mainly via the clathrin-mediated pathway, whereas H-Plate and H-Rod were internalized into the cells mainly via macropinocytosis. The endocytosed nano-HAP was mainly distributed in the cell lysosome. The adhesion and endocytosis of HAP to A7R5 cells were positively correlated with the specific surface area, and contact area of HAP and negatively correlated with the absolute value of Zeta and contact angle of HAP. This study provided insights into the effect of crystal morphology on vascular calcification and its mechanism., (© 2019 Wiley Periodicals, Inc.)

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process., (© 2019 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.)

Atherosclerosis is a chronic arterial disease characterized by vascular inflammation, accumulation of lipids in the arterial wall, and formation and growth of atherosclerotic plaques followed by ischemia. In subclinical atherosclerosis, cholesterol retention in subendothelial cells leads to induction of local inflammation, generation of foam cells and lesion formation, followed by a chain of other pathogenic events. Atherosclerotic progression can frequently be fatal, since plaque rupture may lead to thrombosis and acute events, such as myocardial infarction, stroke and sudden death. Traditional anti-atherosclerotic therapy is mainly focused on improving the blood lipid profile and does not target various stages of plaque progression. Obviously, treating the disease at initial stages is better than beginning treatment at advanced stages and, in that regard, current atherosclerosis management can be improved. Cholesterol retention is an important component of atherogenesis that precedes plaque formation. Therapeutic targeting of cholesterol retention may be beneficial for preventing further atherogenic progression. For this purpose, we suggest using herbal preparations due to good tolerability and suitability for long-lasting treatment. We developed test systems based on cultured human intimal aortic cells for rapid screening of potential anti-atherogenic drugs. With the help of these test systems, we selected several natural substances with significant anti-atherogenic activity and further use these compounds to prepare herbal preparations for anti-atherosclerotic therapy. These preparations were clinically tested and showed good safety and a potent anti-atherogenic potential., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Metallothionein (MT) is an inducible protein with cytoprotective activity against heavy metals such as cadmium, zinc, and copper. MT-1 and MT-2 are the isoforms of MT induced by and bind the heavy metals. Bovine aortic endothelial cells contain three types of MT genes, namely, MT-1A, MT-1E, and MT-2A; however, the associated protein expression of these MT isoforms has not been identified. In the present study, the expression of MT subisoform proteins in cells treated with cadmium chloride was identified using a high-performance liquid chromatography-inductively coupled plasma-mass spectrometry system. It was revealed that: (1) transcriptional induction of MT-1A by cadmium was markedly more sensitive than that of MT-1E/2A; (2) MT-1A and MT-2A proteins were the predominant MT subisoforms induced by cadmium; and (3) there might be differentiation in the functions of MT-1 and MT-2 against cadmium cytotoxicity, although the actual roles of the MT isoforms in the cells were not distinct. This is the first study to show the differential induction of isoforms of MT proteins in vascular endothelial cells by cadmium.

Premature senescence, a death escaping pathway for cells experiencing stress, is conducive to aging and cardiovascular diseases. The molecular switch between senescent and apoptotic fate remains, however, poorly recognized. Nrf2 is an important transcription factor orchestrating adaptive response to cellular stress. Here, we show that both human primary endothelial cells (ECs) and murine aortas lacking Nrf2 signaling are senescent but unexpectedly do not encounter damaging oxidative stress. Instead, they exhibit markedly increased S-nitrosation of proteins. A functional role of S-nitrosation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage and redirection of ECs to premature senescence. S-nitrosation and senescence are mediated by Keap1, a direct binding partner of Nrf2, which colocalizes and precipitates with nitric oxide synthase (NOS) and transnitrosating protein GAPDH in ECs devoid of Nrf2. We conclude that the overabundance of this "unrestrained" Keap1 determines the fate of ECs by regulation of S-nitrosation and propose that Keap1/GAPDH/NOS complex may serve as an enzymatic machinery for S-nitrosation in mammalian cells., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

It has been shown that anti-inflammatory cytokines interleukin-35 (IL-35) and IL-10 could inhibit acute endothelial cell (EC) activation, however, it remains unknown if and by what pathways IL-35 and IL-10 could block atherogenic lipid lysophosphatidylcholine (LPC)-induced sustained EC activation; and if mitochondrial reactive oxygen species (mtROS) can differentiate mediation of EC activation from trained immunity (innate immune memory). Using RNA sequencing analyses, biochemical assays, as well as database mining approaches, we compared the effects of IL-35 and IL-10 in LPC-treated human aortic ECs (HAECs). Principal component analysis revealed that both IL-35 and IL-10 could similarly and partially reverse global transcriptome changes induced by LPC. Gene set enrichment analyses showed that while IL-35 and IL-10 could both block acute EC activation, characterized by upregulation of cytokines/chemokines and adhesion molecules, IL-35 is more potent than IL-10 in suppressing innate immune signatures upregulated by LPC. Surprisingly, LPC did not induce the expression of trained tolerance itaconate pathway enzymes but induced trained immunity enzyme expressions; and neither IL-35 nor IL-10 was found to affect LPC-induced trained immunity gene signatures. Mechanistically, IL-35 and IL-10 could suppress mtROS, which partially mediate LPC-induced EC activation and innate immune response. Therefore, anti-inflammatory cytokines could reverse mtROS-mediated acute and innate immune trans-differentiation responses in HAECs, but it could spare metabolic reprogramming and trained immunity signatures, which may not fully depend on mtROS. Our characterizations of anti-inflammatory cytokines in blocking mtROS-mediated acute and prolonged EC activation, and sparing trained immunity are significant for designing novel strategies for treating cardiovascular diseases, other inflammatory diseases, and cancers., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Currently, there is no doubt surrounding a theory that the cardiotropic effects of sex hormones can be due to their direct effect on the cardiovascular system. In recent years, interest in the study of steroid glycosides has increased. We studied the effects of furostanol glycosides (protodioscin and deltozid) from the cell culture of the Dioscorea deltoidea (laboratory code DM-05) on the physiological and biochemical parameters of vascular endothelial function in hypoestrogen-induced endothelial dysfunction after bilateral ovariectomy. It was shown that the use of DM-05 at a dose of 1 mg/kg makes it possible to prevent the development of arterial hypertension (the level of systolic blood pressure (SBP) decreases by 9.7% ( p < 0.05) and diastolic blood pressure (DBP) by 8.2%), to achieve a decrease in the coefficient of endothelial dysfunction by 1.75 times against the background of a hypoestrogenic state. With DM-05, an increase in the concentration of stable nitric oxide metabolites (NO x ) by 45.6% ( p < 0.05) and an increase in mRNA endothelial nitric oxide synthase (eNOS) expression by 34.8% ( p < 0.05) was established, which indicates a positive effect of furostanol glycosides on the metabolism of nitric oxide after ovariectomy. Positive dynamics in the histological structure of the heart and the abdominal aorta indicate the pronounced endothelio- and atheroprotective effects of DM-05., Competing Interests: The authors declare no conflicts of interest.

A major limitation in extrusion-based bioprinting is the lack of direct process control, which limits the accuracy and design complexity of printed constructs. The lack of direct process control results in a number of defects that can influence the functional and mechanical outcomes of the fabricated structures. The machine axes motion cannot be reliably used to predict material placement, and precise fabrication requires additional sensing of the material extrusion. We present an iteration-to-iteration process monitoring system that enables direct process control in the material deposition reference frame. To fabricate parts with low dimensional errors, we integrate a non-contact laser displacement scanner into the printing platform. After fabrication of the initial print using the as-designed reference trajectory, the laser scanner moves across the part to measure the material placement. A custom image processing algorithm compares the laser scanner data to the as-designed reference trajectory to generate an error vector. To compensate for the measured error, the algorithm modifies the axes reference trajectory for the second print iteration. We implement the in situ process monitoring and error compensation technique on an experimental platform to evaluate system performance and demonstrate improvement in spatial material placement.

Vascular smooth muscle cells (VSMCs) of ascending aorta and TBX18 + sinus node both originated from the second heart field. The study explored whether ascending aortic smooth muscle cells in vitro could be reprogrammed into pacemaker-like cells with human TBX18 . In the study, VSMCs were infected with TBX18 , and then cocultured with neonatal rat ventricular cardiomyocytes (NRVMs) in vitro . By overexpressing TBX18 , the transfected VSMCs expressed high levels of hyperpolarization-activated cyclic nucleotide-gated channel 4 ( HCN4 ), insulin gene enhancer binding protein 1, and human dwarf homeobox gene SHOX2, cardiac troponin I, and low level of connexin 43. In addition, funny current (I f ) was recorded by patch clamp appeared the time and voltage dependence in TBX18 group, which the amplitude of I f density was from - 5.164 ± 0.662 pA/pF to -0.765 ± 0.358 pA/pF ( n  = 14). Furthermore, TBX18 -transfected VSMCs coupled with NRVMs showed typical action potential of pacemaker-like cells and the beating rate was faster (178.00 ± 7.55 bpm, p  < 0.05) compared with other groups. In conclusion, our study indicated that transcription factor TBX18 could reprogram VSMCs into pacemaker-like cells in vitro .

Poly-l-lactic acid (PLLA) is widely used in clinic, for example, as biodegradable coronary artery stents. However, inflammatory responses in endothelial cells associated with PLLA degradation are relatively undefined. We previously reported inflammation in human aortic endothelial cells (HAEC) in vitro and in vivo. Here, we further assessed inflammatory injury, including cell migration, cell function, and inflammatory cytokines expressed in HAEC treated with PLLA and curcumin by CCK-8, wound healing assay, ELISA, and Western blot. Significant inhibition of cell migration, remarkable dysfunction, and inflammatory responses were found in HAEC treated with PLLA degradation extract, and these effects were alleviated by Cur treatment. These findings indicated that cautious evaluation of biodegradable polymers should be performed, and Cur represents a promising anti-inflammatory agent for alleviating endothelial dysfunction and inflammation caused by PLLA degradation. In addition, Cur should be further studied experimentally in in vivo experiments on animal models as a potential therapeutic to reduce thrombosis of biodegradable polymer stents., (© 2019 Wiley Periodicals, Inc.)

Decellularized porcine aorta (PA) is a promising biomaterial for vascular substitutes. However, decellularized PAs suffer from mechanical weakness and have less pores, which limit cellular ingrowth into the grafts and hinder the remodeling. In this study, PAs were decellularized by vacuum-freeze-thawing cycles and 0.3% of sodium dodecyl sulfate (SDS) buffer (VLS). Results showed that the application of vacuum-freeze-thawing significantly improved the decellularization efficiency of SDS while effectively preserved the mechanical function of PA tissues, decreased residual SDS, and minimized cytotoxicity. Furthermore, scanning electron microscopy (SEM) examination demonstrated that VLS generated interconnected pores with uniform distribution. In vivo subcutaneous implantation assay further demonstrated that VLS implants had less calcification and adverse inflammatory response. Moreover, VLS treatment markedly enhanced ingrowth of myofibroblasts and endothelial cells, and thereby promoted synthesis of extracellular matrix and vascularization. These results suggest that the application of vacuum-freeze-thawing into the decellularization process may produce a promising vascular graft candidate for tissue engineering application., (© 2019 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases., (© 2019 Wiley Periodicals, Inc.)

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the development and progression of diabetes-related vascular complications. Recently, microRNAs (miRNAs) have been suggested to be involved in the pathogenesis of vascular diseases. This study was designed to investigate the influences of tanshinone IIA, an active compound extracted from Chinese herb Salvia miltiorrhiza, on the proliferation and migration of human aortic VSMCs (HASMCs). cultured in a high glucose medium and the underlying mechanisms related miRNAs. Using a miRNA microarray method, we profiled the miRNA expression signature in human aortic VSMCs (HASMCs) exposed to normal glucose, high glucose with and without Tanshinone IIA. Cell proliferation was measured with 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell migration was evaluated using transwell migration assay and wound scratch assay. Western blot was used to examine the expression of tropomyosin 1 (TPM1) and miRNA level was quantified by real-time PCR. The results showed that several miRNAs that were highly expressed in the high glucose group were significantly decreased in the high glucose with Tanshinone IIA group compared with the normal glucose group (P < 0.05). Among these miRNAs, miR-21-5p was significantly upregulated in the high glucose group and downregulated after Tanshinone IIA treatment (P < 0.05). The depletion of miR-21-5p in HASMCs resulted in decreased cell proliferation and migration (P < 0.05). Moreover, we found that Tanshinone IIA inhibited proliferation and migration partly through miR-21-5p-mediated TPM1 downregulation (P < 0.05). In conclusion, the present study demonstrates that Tanshinone IIA is able to protect HASMCs from high glucose-induced proliferation and migration through regulating expression of miRNAs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea leaves, has anti-inflammatory effects. In this study, we investigated the mechanism by which EGCG attenuates the effects of lipopolysaccharide (LPS), an agonist of toll-like receptor 4 (TLR4), in cultured human aortic endothelial cells (HAECs). The increase in the expression of intercellular adhesion molecule-1 (ICAM-1) induced by LPS (100 ng/ml) was effectively attenuated by pretreatment with EGCG (50 μM). Importantly, EGCG treatment resulted in a rapid reduction of cellular TLR4, which was accompanied by an increase in the N-terminal fragment of TLR4 in the culture supernatant, indicating that EGCG induces ectodomain shedding of TLR4. EGCG increased cytosolic Ca 2+ by inducing the release of intracellular stored Ca 2+ and the influx of extracellular Ca 2+ ; accordingly, EGCG-induced ectodomain shedding of TLR4 was nullified by pretreatment with BAPTA-AM (10 μM), an intracellular Ca 2+ chelator. EGCG induced translocation of a disintegrin and metalloprotease 10 (ADAM10) to the cell surface, which was also blocked by BAPTA-AM. Treatment with ADAM10 inhibitor (GI254023X, 2 μM) and siRNA-mediated depletion of ADAM10 prevented EGCG-induced ectodomain shedding of TLR4 and abolished the inhibitory effect of EGCG on LPS-induced ICAM-1 expression. Collectively, these findings suggest that EGCG decreases cell surface TLR4 in HAECs by inducing ADAM10-mediated ectodomain shedding, and thereby attenuates the effects of LPS. This is a new mechanism of the suppressive effect of EGCG on LPS signaling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications.

Endothelial dysfunction is considered to be an initial indicator in diabetes-induced macrovascular complications. Evidence has shown that CGRP is an important neuropeptide active in vascular system, especially in vasorelaxation. This study aimed to investigate the role of CGRP in high-glucose-induced endothelial dysfunction in rat aorta endothelial cells (RAECs). Quantitative-real time PCR and western blots were used to determine the efficiency of overexpression and interference of CGRP. After incubation with normal glucose (5.5 mM) or high glucose (33 mM), the cell viability and cell apoptosis were tested. Afterwards, the Nitric Oxide (NO) production, the mRNA expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and angiotensin II (Ang II) and the level of reactive oxygen species (ROS) were determined. The involvement of ERK1/2-NOX4 was determined through western blots and the translocation of p47phox was also observed via cell immunofluorescence. CGRP alleviated the high-glucose-induced cell apoptosis while CGRP did not have an obvious impact on cell viability. Meanwhile, CGRP increased the NO production as well as the eNOS mRNA expression and reversely decreased the stimulated expression of iNOS and Ang II by high glucose. In addition, CGRP attenuated the high-glucose-stimulated intracellular ROS production by ERK1/2-NOX4 and the translocation of p47phox. These results indicated the protective role of CGRP in high-glucose-induced oxidative injury in RAECs possibly through inhibiting ERK1/2-NOX4. Our findings might help to further understand the potential role and possible mechanism of CGRP in endothelial dysfunction caused by high glucose., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Cardiovascular disease is the leading cause of death in patients with chronic kidney disease, so there is an urgent need to identify therapeutic targets to control the progression of cardiovascular disease. Apoptosis of aortic smooth muscle cells can promote cardiovascular disease, but the role of parathyroid hormone (PTH) and sirtuin 1 in the pathophysiology of apoptosis is still unclear., Methods: Cultured human aortic smooth muscle cells (HASMCs) were stimulated with 10 -6 , 10 -8 , or 10 -10  mol/L PTH for different days, apoptosis was measured by flow cytometry and sirtuin 1 and Bcl-2 protein levels in cell extracts were analyzed by western blotting. HASMCs were stimulated with PTH (10 -8  mol/L) and 50 or 100 μmol/L RES for 3 d, apoptosis was measured by flow cytometry and sirtuin 1 and Bcl-2 protein levels in cell extracts were analyzed by western blotting., Results: We found that PTH decreased the expression of sirtuin 1 and Bcl-2, inducing apoptosis (p<.05). Resveratrol (RES), a sirtuin 1 agonist, inhibited PTH-induced apoptosis and restored Bcl-2 expression (p<.05)., Conclusions: PTH induces apoptosis in HASMCs. Resveratrol inhibits PTH-induced apoptosis in HASMCs.

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.

Voltage-dependent L-type Ca 2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 ( P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction ( P < 0.05). Changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca 2+ ] i in A7r5 cells was suppressed by 1-10 µM Y-27632 ( P < 0.05), but 10 µM Y-27632 was required to suppress Ca 2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca 2+ ] i . Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca 2+ ] i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and β-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho., (© 2019 The Author(s).)

Mast cells play an important role in immunomodulation and in the maintenance of vascular integrity. Interleukin-6 (IL-6) is one of the key biomarkers and therapeutic target in systemic vasculitis. The objective of the current study is to describe the role of mast cells in arterial IL-6 homeostasis. Eight- to ten-week-old male C57BL/6 (wild-type) mice were injected with either (a) saline, (b) compound 48/80 (a systemic mast cell degranulating agent), (c) lipopolysaccharide (LPS), or (d) a combination of C48/80 and LPS. Twenty-four hours after the injections, mice were sacrificed and serum samples and aortic tissues were analyzed for determining inflammatory response and cytokine expression profile. The results revealed that induction of mast cell degranulation significantly lowers serum IL-6 levels and aortic expression of IL-6 in LPS-treated mice. Significantly higher aortic expression of toll-like receptor-2 (TLR-2) and TNF- α was seen in the LPS and LPS+C48/80 groups of mice compared to controls. Aortic expression of TLR-4 was significantly decreased in LPS+C48/80 compared to C48/80 alone. LPS+C48/80-treated mice presented with a 3-fold higher aortic expression of suppressor of cytokine signaling (SOCS-1) compared to saline-injected groups. The inhibition of LPS-induced increase in serum IL-6 levels by mast cell degranulation was not seen in H1R knockout mice which suggests that mast cell-derived histamine acting through H1R may participate in the regulatory process. To examine whether the mast cell-mediated downregulation of LPS-induced IL-6 production is transient or cumulative in nature, wild-type mice were injected serially over a period of 10 days (5 injections) and serum cytokine levels were quantified. We found no significant differences in serum IL-6 levels between any of the groups. While mice injected with C48/80 or LPS had higher IL-10 compared to vehicle-injected mice, there was no difference between C48/80- and LPS+C48/80-injected mice. In conclusion, in an in vivo setting, mast cells appear to partially and transiently regulate systemic IL-6 homeostasis. This effect may be regulated through increased systemic IL-10 and/or aortic overexpression of SOCS-1., Competing Interests: All the authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Jason M. Springer et al.)

Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-β by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-β levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-β has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-β turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)