Vascular Cell Activation Research Articles

Antioxidant peptides derived from hydrolysates of red tilapia (Oreochromis sp.) scale

Processing red tilapia (Oreochromis sp.) yields a large quantity of scale, a byproduct of the aquaculture industry. This study focuses on evaluating the potential of red tilapia scales as a source of antioxidant peptides. Scales were ground and enzymatically hydrolyzed by Alcalase® 2.4 L. The hydrolysate obtained was fractionated to purify and identify the antioxidant peptides by membrane ultrafiltration and chromatographic technology guided by the antioxidant activity in rat vascular smooth muscle A7r5 cells. After ultrafiltration, the fraction with molecular weight of 3–10 kDa showed the highest activity and their chemical nature was hydrophilic. The sequence of twenty antioxidant peptides, containing 6–16 amino acids, was identified by liquid chromatography–tandem mass spectrometry. The results indicate that the red tilapia (Oreochromis sp.) scale could be a new natural source of antioxidant peptides.

Mechanisms Contributing to the Dysregulation of miRNA-124 in Pulmonary Hypertension.

Chronic pulmonary hypertension (PH) is a fatal disease characterized by the persistent activation of pulmonary vascular cells that exhibit aberrant expression of genes including miRNAs. We and others reported that decreased levels of mature microRNA-124 (miR-124) plays an important role in modulating the activated phenotype of pulmonary vascular cells and HDAC inhibitors (HDACi) can restore the levels of mature miR-124 and reverse the persistently activated phenotype of PH vascular cells. In this study, we sought to determine the mechanisms contributing to reduced levels of miRNAs, as well as how HDACi restores the levels of reduced miRNA in PH vascular cells. We found that pulmonary artery fibroblasts isolated from IPAH patients (PH-Fibs) exhibit reduced levels of mature miR-124 and several other miRNAs including let-7i, miR-224, and miR-210, and that these reduced levels can be restored by HDACi. Using miR-124 expression in human PH-Fibs as a model, we determined that reduced miR-124 gene transcription, not decreased expression of miRNA processing genes, is responsible for reduced levels of mature miR-124 in human PH-Fibs. Using both DNase I Sensitivity and chromatin immunoprecipitation assays, we found that the miR-124-1 gene exhibits a more condensed chromatin structure in human PH-Fibs, compared to corresponding controls. HDACi relaxed miR-124-1 chromatin structure, evidenced by increased levels of the open chromatin mark H3K27Ac, but decreased levels of closed chromatin mark H3K27Me3. Most importantly, the delivery of histone acetyltransferase (HAT) via CRISPR-dCas9-HAT and guiding RNAs to the promoter of the miR-124-1 gene increased miR-124-1 gene transcription. Thus, our data indicate epigenetic events play important role in controlling miR-124 and likely other miRNA levels and epigenetic regulators such as HDACs appear to be promising therapeutic targets for chronic PH.

GENETIC SUSCEPTIBILITY TO EARLY ONSET OF TRANSFORMING GROWTH FACTOR-BETA DRIVEN CARDIAC REMODELING IN MICE IS ASSOCIATED WITH EGR1 AND STAT3

Objective: We have previously described that the severity of TGF-b induced cardiac remodeling was associated with genetic background differences in alb/TGF-b1 transgenic mice (with elevated plasma TGF-b1 levels) on B6 vs CBAxB6 strains. Early growth response factor-1 (EGR1) has been associated with kidney fibrosis, and we recently demonstrated non-canonical TGF-b induced STAT3 activation in vascular smooth muscle cells, but their role in cardiac fibrosis or hypertrophy was not yet elucidated. The present study investigated whether genetic susceptibility to TGF-b induced early cardiac remodeling is associated with transcription factors EGR1 and STAT3. Design and method: As the albumin promoter driven TGF-b1 transgene is expressed after birth only in males reaching early high plasma TGF-b1 levels, we investigated 14-day old male transgenic and wild type control mice. Blood pressure of TGF-b1 transgenic (B6-TGFb and CBAxB6-TGFb) mice, and wild type (B6 and CBAxB6) controls (n = 6–9/group) was assessed, then cardiac samples were harvested and analyzed for mRNA and protein expression. Data are presented as mean ± SD and evaluated using Kruskal-Wallis test with Dunn's post-hoc test. Results: Blood pressure was elevated in both transgenic strains as compared to controls (MAP mmHg, B6: 46 ± 3; CBAxB6: 48 ± 4; B6-TGFb:54 ± 9; CBAxB6-TGFb:68 ± 7, p < 0.01). Compared to B6-TGFb and wild type, CBAxB6-TGFb hearts showed 20-fold elevation in BNP mRNA relative expression (B6: 1.5 ± 0.9; CBAxB6: 2.3 ± 0.9; B6-TGFb:1.5 ± 1.0; CBAxB6-TGFb:23.5 ± 13,3 p < 0.001) associated with increased Collagen-I (B6: 1.3 ± 0.2; CBAxB6: 1.2 ± 0.1; B6-TGFb:1.2 ± 0.2; CBAxB6-TGFb:1.9 ± 0.6, p < 0.05) and EGR1 (B6: 0.9 ± 0.2; CBAxB6: 0.8 ± 0.3; B6-TGFb:0.9 ± 0.6; CBAxB6-TGFb:1.8 ± 0.5, p < 0.01) as well as STAT3 mRNA overexpression (B6: 0.8 ± 0.1; CBAxB6: 1.2 ± 0.2; B6-TGFb:1.1 ± 0.1; CBAxB6-TGFb:1.7 ± 0.6, p < 0.01) and STAT3 phosphorylation. Early cardiac remodeling was further associated with 12-fold and 2-fold overexpression of TIMP-1 (B6: 1.0 ± 0.4; CBAxB6: 0.8 ± 0.4; B6-TGFb:0.7 ± 0.3; CBAxB6-TGFb:12.2 ± 9.0, p < 0.01) and TIMP-2 (B6: 1.1 ± 0.1; CBAxB6: 1.2 ± 0.2; B6-TGFb:1.3 ± 0.4; CBAxB6-TGFb:2.1 ± 1.1, p < 0.05), respectively. Significant EGR1 immunoreactivity was detected only in cardiomyocytes of CBAxB6-TGFb hearts. Conclusions: We conclude that EGR1 and STAT3 might strongly contribute to the initiation and early progression of cardiac remodeling driven by high circulating TGF-b levels. We postulate that EGR1 and STAT3 could be potential targets to inhibit early cardiac remodeling.

Transforming growth factor‐β1 inhibits interleukin‐1β‐induced expression of inflammatory genes and Cathepsin S activity in human vascular smooth muscle cells

This study investigated the opposite mechanisms by which IL-1β and TGF-β1 modulated the inflammatory and migratory phenotypes in cultured human intimal vascular smooth muscle cells vSMCs. Primary human vSMCs, obtained from twelve hypertensive patients who underwent carotid endarterectomy, were incubated for 24hours with either 40pM TGF-β1, or 1nmol/L IL-1β, or their combination in presence or absence of anti-TGF-β neutralizing antibody. The expression levels of matrix metalloproteases and their inhibitors, and the elastolytic enzyme cathepsin S (CTSS) and its inhibitor cystatin C were evaluated with RT-PCR. CTSS activity was measured by fluorometry. TGF-β1 reversed IL-1β-induced expression of iNOS, CXCL6, IL1R1, MMP12, and CTSS, while upregulated TIMP2 expression. Furthermore, anti-TGF-β neutralizing antibody abrogated TGF-β effects. Combination with IL-1β and TGF-β1 induced the expression of IL1α, IL1β, IL1R1, and CTSS, but suppressed CST3 expression. CTSS expression in the combination treatment was higher than that of cells treated with anti-TGF-β antibodies alone. Moreover, IL-1β-induced CTSS enzymatic activity was reduced when human vSMCs were co-treated with TGF-β, whereas this reduction was abrogated by anti-TGF-β neutralizing antibody. TGF-β1 abrogated IL-1β-induced expression of inflammatory genes and elastolytic activity in cultured human vSMCs. Thus, TGF-β1 can play a crucial role in impairing IL-1β-induced vascular inflammation and damage involved in the etiology of cardiovascular diseases.

Chronic Kidney Disease: Mitochondrial Damage‐Induced Innate Immune Activation in Vascular Smooth Muscle Cells Promotes Chronic Kidney Disease‐Associated Plaque Vulnerability (Adv. Sci. 5/2021)

Chronic Kidney Disease: Mitochondrial Damage‐Induced Innate Immune Activation in Vascular Smooth Muscle Cells Promotes Chronic Kidney Disease‐Associated Plaque Vulnerability (Adv. Sci. 5/2021)

Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19

Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.

Spent Hen Muscle Protein-Derived RAS Regulating Peptides Show Antioxidant Activity in Vascular Cells.

Spent hens are egg-laying hens reaching the end of their egg-laying cycles, being a major byproduct of the egg industry. Recent studies have been focusing on finding new value-added uses for spent hens. We have previously identified four bioactive peptides from spent hen muscle proteins, including three angiotensin-converting enzyme (ACE) inhibitory (ACEi) peptides (VRP, LKY, and VRY), and one ACE2 upregulating (ACE2u) peptide (VVHPKESF (V-F)). In the current study, we further assessed their antioxidant and cytoprotective activities in two vascular cell lines—vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs)—upon stimulation by tumor necrosis factor alpha (TNFα) and angiotensin (Ang) II, respectively. The results from our study revealed that all four peptides attenuated oxidative stress in both cells. None of the investigated peptides altered the expression of TNFα receptors in ECs; however, VRY and V-F downregulated Ang II type 1 receptor (AT1R), while V-F upregulated the Mas receptor (MasR) in VSMCs. Further, we found that the antioxidant effects of VRP, LKY, and VRY were likely through acting as direct radical scavengers, while that of V-F was at least partially ascribed to increased endogenous antioxidant enzymes (GPx4 and SOD2) in both cells. Besides, as an ACE2u peptide, V-F exerted antioxidant effect in a MasR-dependent manner, indicating a possible involvement of the upregulated ACE2-MasR axis underlying its antioxidant action. The antioxidant activities of VRP, LKY, VRY, and V-F in vascular cells indicated their multifunctional properties, in addition to their ACEi or ACE2u activity, which supports their potential use as functional food ingredients against hypertension.

Short Chain (≤C4) Esterification Increases Bioavailability of Rosmarinic Acid and Its Potency to Inhibit Vascular Smooth Muscle Cell Proliferation

Rosmarinic acid is a natural phenolic acid and active compound found in many culinary plants, such as rosemary, mint, basil and perilla. Aiming to improve the pharmacokinetic profile of rosmarinic acid and its activity on vascular smooth muscle cell proliferation, we generated a series of rosmarinic acid esters with increasing alkyl chain length ranging from C1 to C12. UHPLC-MS/MS analysis of rat blood samples revealed the highest increase in bioavailability of rosmarinic acid, up to 10.52%, after oral administration of its butyl ester, compared to only 1.57% after rosmarinic acid had been administered in its original form. When added to vascular smooth muscle cells in vitro, all rosmarinic acid esters were taken up, remained esterified and inhibited vascular smooth muscle cell proliferation with IC50 values declining as the length of alkyl chains increased up to C4, with an IC50 of 2.84 µM for rosmarinic acid butyl ester, as evident in a resazurin assay. Vascular smooth muscle cells were arrested in the G0/G1 phase of the cell cycle and the retinoblastoma protein phosphorylation was blocked. Esterification with longer alkyl chains did not improve absorption and resulted in cytotoxicity in in vitro settings. In this study, we proved that esterification with proper length of alkyl chains (C1–C4) is a promising way to improve in vivo bioavailability of rosmarinic acid in rats and in vitro biological activity in rat vascular smooth muscle cells.

Mitochondrial Damage-Induced Innate Immune Activation in Vascular Smooth Muscle Cells Promotes Chronic Kidney Disease-Associated Plaque Vulnerability.

Chronic kidney disease (CKD) is associated with accelerated atherosclerosis progression and high incidence of cardiovascular events, hinting that atherosclerotic plaques in CKD may be vulnerable. However, its cause and mechanism remain obscure. Here, it is shown that apolipoprotein E‐deficient (ApoE−/−) mouse with CKD (CKD/ApoE−/− mouse) is a useful model for investigating the pathogenesis of plaque vulnerability, and premature senescence and phenotypic switching of vascular smooth muscle cells (VSMCs) contributes to CKD‐associated plaque vulnerability. Subsequently, VSMC phenotypes in patients with CKD and CKD/ApoE−/− mice are comprehensively investigated. Using multi‐omics analysis and targeted and VSMC‐specific gene knockout mice, VSMCs are identified as both type‐I‐interferon (IFN‐I)‐responsive and IFN‐I‐productive cells. Mechanistically, mitochondrial damage resulting from CKD‐induced oxidative stress primes the cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) pathway to trigger IFN‐I response in VSMCs. Enhanced IFN‐I response then induces VSMC premature senescence and phenotypic switching in an autocrine/paracrine manner, resulting in the loss of fibrous cap VSMCs and fibrous cap thinning. Conversely, blocking IFN‐I response remarkably attenuates CKD‐associated plaque vulnerability. These findings reveal that IFN‐I response in VSMCs through immune sensing of mitochondrial damage is essential for the pathogenesis of CKD‐associated plaque vulnerability. Mitigating IFN‐I response may hold promise for the treatment of CKD‐associated cardiovascular diseases.

TGF-β loaded exosome enhances ischemic wound healing in vitro and in vivo.

Rationale: With over seven million infections and $25 billion treatment cost, chronic ischemic wounds are one of the most serious complications in the United States. The controlled release of bioactive factor enriched exosome from finbrin gel was a promising strategy to promote wound healing.Methods: To address this unsolved problem, we developed clinical-grade platelets exosome product (PEP), which was incorporate with injectable surgical fibrin sealant (TISSEEL), to promote chronic wound healing and complete skin regeneration. The PEP characterization stimulated cellular activities and in vivo rabbit ischemic wound healing capacity of TISSEEL-PEP were performed and analyzed.Results: PEP, enriched with transforming growth factor beta (TGF-β), possessed exosomal characteristics including exosome size, morphology, and typical markers including CD63, CD9, and ALG-2-interacting protein X (Alix). In vitro, PEP significantly promoted cell proliferation, migration, tube formation, as well as skin organoid formation. Topical treatment of ischemic wounds with TISSEEL-PEP promoted full-thickness healing with the reacquisition of hair follicles and sebaceous glands. Superior to untreated and TISSEEL-only treated controls, TISSEEL-PEP drove cutaneous healing associated with collagen synthesis and restoration of dermal architecture. Furthermore, PEP promoted epithelial and vascular cell activity enhancing angiogenesis to restore blood flow and mature skin function. Transcriptome deconvolution of TISSEEL-PEP versus TISSEEL-only treated wounds prioritized regenerative pathways encompassing neovascularization, matrix remodeling and tissue growth.Conclusion: This room-temperature stable, lyophilized exosome product is thus capable of delivering a bioactive transforming growth factor beta to drive regenerative events.

Designed CXCR4 mimic acts as a soluble chemokine receptor that blocks atherogenic inflammation by agonist-specific targeting

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides (‘msR4Ms’) designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe−/− mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.

Eosinophils Protect Mice From Angiotensin-II Perfusion-Induced Abdominal Aortic Aneurysm.

Blood eosinophil count and ECP (eosinophil cationic protein) associate with human cardiovascular diseases. Yet, whether eosinophils play a role in cardiovascular disease remains untested. The current study detected eosinophil accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting eosinophil participation in this aortic disease. To test whether and how eosinophils affect AAA growth. Population-based randomized clinically controlled screening trials revealed higher blood eosinophil count in 579 male patients with AAA than in 5063 non-AAA control (0.236±0.182 versus 0.211±0.154, 109/L, P<0.001). Univariate (odds ratio, 1.381, P<0.001) and multivariate (odds ratio, 1.237, P=0.031) logistic regression analyses indicated that increased blood eosinophil count in patients with AAA served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected eosinophil accumulation and eosinophil cationic protein expression in human and murine AAA lesions. Results showed that eosinophil deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell loss using angiotensin-II perfusion-induced AAA in Apoe-/- and eosinophil-deficient Apoe-/-ΔdblGATA mice. Eosinophil deficiency increased lesion chemokine expression, muted lesion expression of IL (interleukin) 4 and eosinophil-associated-ribonuclease-1 (mEar1 [mouse EOS-associated-ribonuclease-1], human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, eosinophil-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b+Ly6Chi monocytes, and increased CD11b+Ly6Clo monocytes. mEar1 treatment or adoptive transfer of eosinophil from wild-type and Il13-/- mice, but not eosinophil from Il4-/- mice, blocked AAA growth in Apoe-/-ΔdblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for eosinophil IL4 and mEar1 in blocking NF-κB (nuclear factor-κB) activation in macrophages, smooth muscle cells, and endothelial cells. Eosinophils play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.

Mitigative Effect of Sipjeondaebo-tang on RhoA Activation in Cold-Exposed Vascular Cells

Objective To investigate the vasodilatory effect of Sipjeondaebo-tang by inhibiting RhoA activity in vascular cells during cold exposure. Methods Human vascular endothelial cells and pericytes were pretreated with Sipjeondaebo-tang for 30 min, followed by incubation at 37 °C (control) or 25 °C (cold exposure) for 30 min. Activation of endothelin-1-mediated RhoA in pericytes was assessed by pretreating the cells with Sipjeondaebo-tang for 30 min, followed by incubation with endothelin-1 at 37 °C for 30 min. Western blotting was performed to measure the expression of active RhoA. Endothelin-1 and nitric oxide release from endothelial cells was examined with enzyme-linked immunosorbent assay kits. The formation of stress fibers and focal adhesion complexes was analyzed by immunocytochemistry. Results Cold treatment activated RhoA in both pericytes and vascular endothelial cells, whereas Sipjeondaebo-tang treatment inhibited this activation. Sipjeondaebo-tang treatment also reversed the cold-mediated production of endothelin-1 and nitric oxide. Cold exposure promoted the formation of stress fibers and focal adhesion complexes by increasing the expression of phospho-focal adhesion complex kinase, whereas Sipjeondaebo-tang treatment suppressed this response. Conclusions These findings suggested that Sipjeondaebo-tang inhibits cold-induced RhoA activation and its related pathway components, including endothelin-1 and nitric oxide, in vascular cells. Therefore, Sipjeondaebo-tang could be beneficial for the treatment of Raynaud’s phenomenon. Keywords: Raynaud’s phenomenon, cold, rhoA, endothelin-1, nitric oxide, Sipjeondaebo-tang

Targeting Heme Oxygenase-1 in the Arterial Response to Injury and Disease.

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme into carbon monoxide (CO), iron, and biliverdin, which is rapidly metabolized to bilirubin. The activation of vascular smooth muscle cells (SMCs) plays a critical role in mediating the aberrant arterial response to injury and a number of vascular diseases. Pharmacological induction or gene transfer of HO-1 improves arterial remodeling in animal models of post-angioplasty restenosis, vascular access failure, atherosclerosis, transplant arteriosclerosis, vein grafting, and pulmonary arterial hypertension, whereas genetic loss of HO-1 exacerbates the remodeling response. The vasoprotection evoked by HO-1 is largely ascribed to the generation of CO and/or the bile pigments, biliverdin and bilirubin, which exert potent antioxidant and anti-inflammatory effects. In addition, these molecules inhibit vascular SMC proliferation, migration, apoptosis, and phenotypic switching. Several therapeutic strategies are currently being pursued that may allow for the targeting of HO-1 in arterial remodeling in various pathologies, including the use of gene delivery approaches, the development of novel inducers of the enzyme, and the administration of unique formulations of CO and bilirubin.

Mdivi-1 attenuates oxidative stress and exerts vascular protection in ischemic/hypoxic injury by a mechanism independent of Drp1 GTPase activity

Vascular dysfunctions such as vascular hyporeactivity following ischemic/hypoxic injury are a major cause of death in injured patients. In this study, we showed that treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of dynamin-related protein 1 (Drp1), significantly improved vascular reactivity in ischemic rats by attenuating oxidative stress. The antioxidative effects of Mdivi-1 were relatively Drp1-independent, and possibly due to an increase in the levels of the antioxidant enzymes, SOD1 and catalase, as well as to enhanced Nrf2 expression. In addition, we found that while Mdivi-1 had little effect on Drp1 GTPase activity in vascular smooth muscle cells, it inhibited hypoxia-induced Drp1 phosphorylation at Ser-616, reducing excessive mitochondrial fission and slightly enhancing mitochondrial fusion. These effects possibly contributed to vascular protection at an early stage of ischemic/hypoxic injury. Finally, Mdivi-1 stabilized hemodynamics, increased vital organ perfusion, and improved rat survival after ischemic/hypoxic injury, proving a promising therapeutic agent for ischemic/hypoxic injury.

The Potential Effect of Glycogen Synthase Kinase-3β Regulating Myocardin, a Transcription Factor Specific to Vascular Smooth Muscle Cells, on Atherosclerosis

Background and Aim: To investigate whether and how inhibition of GSK-3 regulates myocardin activity in the human vascular smooth muscle cells (SMCs), and to explore the action of GSK-3 inhibitor (AR-A014418) on atherosclerosis (AS) in rats. Materials and Methods: The western-blotting and RT-qPCR were conducted to gauging the changes in protein and transcription levels of related factors. Immunoprecipitation was used to discuss the relationship between myocardin and ACTA2. HE staining and red oil staining were performed to demonstrate pathological changes in renal tissue and aorta. Results: AR-A014418 sharply reduced the activity of myocardin and further abated its phosphorylation of serine and threonine and the recruitment of myocardin to ACTA2. Besides, AR-A014418 could alleviate the pathological changes and significantly increase expression levels of ACTA2 in AS rats. Conclusion: GSK-3 may bilaterally regulate the transcriptional activity of myocardin in vascular SMCs. The regulation of GSK-3 on the transcriptional activity of myocardin may be an important mechanism of differentiation and dedifferentiation of vascular smooth muscle and related diseases.

FAK Family Kinases in Vascular Diseases.

In various vascular diseases, extracellular matrix (ECM) and integrin expression are frequently altered, leading to focal adhesion kinase (FAK) or proline-rich tyrosine kinase 2 (Pyk2) activation. In addition to the major roles of FAK and Pyk2 in regulating adhesion dynamics via integrins, recent studies have shown a new role for nuclear FAK in gene regulation in various vascular cells. In particular, FAK primarily localizes within the nuclei of vascular smooth muscle cells (VSMCs) of healthy arteries. However, vessel injury increased FAK localization back to adhesions and elevated FAK activity, leading to VSMC hyperplasia. The study suggested that abnormal FAK or Pyk2 activation in vascular cells may cause pathology in vascular diseases. Here we will review several studies of FAK and Pyk2 associated with integrin signaling in vascular diseases including restenosis, atherosclerosis, heart failure, pulmonary arterial hypertension, aneurysm, and thrombosis. Despite the importance of FAK family kinases in vascular diseases, comprehensive reviews are scarce. Therefore, we summarized animal models involving FAK family kinases in vascular diseases.

FNDC5 Attenuates Oxidative Stress and NLRP3 Inflammasome Activation in Vascular Smooth Muscle Cells via Activating the AMPK-SIRT1 Signal Pathway.

Vascular oxidative stress and inflammation play a major role in vascular diseases. This study was aimed at determining the protective roles of fibronectin type III domain-containing 5 (FNDC5) in angiotensin II- (Ang II-) induced vascular oxidative stress and inflammation and underlying mechanisms. Wild-type (WT) and FNDC5−/− mice, primary mouse vascular smooth muscle cells (VSMCs), and the rat aortic smooth muscle cell line (A7R5) were used in the present study. Subcutaneous infusion of Ang II caused more serious hypertension, vascular remodeling, oxidative stress, NLRP3 inflammasome activation, AMPK phosphorylation inhibition, and SIRT1 downregulation in the aorta of FNDC5−/− mice than those of WT mice. Exogenous FNDC5 attenuated Ang II-induced superoxide generation, NADPH oxidase 2 (NOX2) and NLRP3 upregulation, mature caspase-1, and interleukin-1β (IL-1β) production in A7R5 cells. The protective roles of FNDC5 were prevented by SIRT-1 inhibitor EX527, AMPK inhibitor compound C, or integrin receptor inhibitor GLPG0187. FNDC5 attenuated the Ang II-induced inhibition in SIRT1 activity, SIRT1 protein expression, and AMPKα phosphorylation in A7R5 cells, which were prevented by compound C, EX527, and GLPG0187. FNDC5 deficiency deteriorated Ang II-induced oxidative stress, NLRP3 inflammasome activation, AMPK phosphorylation inhibition, and SIRT1 downregulation in primary aortic VSMCs of mice, which were prevented by exogenous FNDC5. These results indicate that FNDC5 deficiency aggravates while exogenous FNDC5 alleviates the Ang II-induced vascular oxidative stress and NLRP3 inflammasome activation via the AMPK-SIRT1 signal pathway in VSMCs.

ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension.

Human genetics, biomarker, and animal studies implicate loss of function in bone morphogenetic protein (BMP) signaling and maladaptive transforming growth factor-β (TGFβ) signaling as drivers of pulmonary arterial hypertension (PAH). Although sharing common receptors and effectors with BMP/TGFβ, the function of activin and growth and differentiation factor (GDF) ligands in PAH are less well defined. Increased expression of GDF8, GDF11, and activin A was detected in lung lesions from humans with PAH and experimental rodent models of pulmonary hypertension (PH). ACTRIIA-Fc, a potent GDF8/11 and activin ligand trap, was used to test the roles of these ligands in animal and cellular models of PH. By blocking GDF8/11- and activin-mediated SMAD2/3 activation in vascular cells, ACTRIIA-Fc attenuated proliferation of pulmonary arterial smooth muscle cells and pulmonary microvascular endothelial cells. In several experimental models of PH, prophylactic administration of ACTRIIA-Fc markedly improved hemodynamics, right ventricular (RV) hypertrophy, RV function, and arteriolar remodeling. When administered after the establishment of hemodynamically severe PH in a vasculoproliferative model, ACTRIIA-Fc was more effective than vasodilator in attenuating PH and arteriolar remodeling. Potent antiremodeling effects of ACTRIIA-Fc were associated with inhibition of SMAD2/3 activation and downstream transcriptional activity, inhibition of proliferation, and enhancement of apoptosis in the vascular wall. ACTRIIA-Fc reveals an unexpectedly prominent role of GDF8, GDF11, and activin as drivers of pulmonary vascular disease and represents a therapeutic strategy for restoring the balance between SMAD1/5/9 and SMAD2/3 signaling in PAH.

Retinal astrocytes transcriptome reveals Cyp1b1 regulates the expression of genes involved in cell adhesion and migration.

Astrocytes (AC) are the most abundant cells in the central nervous system. In the retina, astrocytes play important roles in the development and integrity of the retinal neurovasculature. Astrocytes dysfunction contributes to pathogenesis of a variety of neurovascular diseases including diabetic retinopathy. Recent studies have demonstrated the expression of Cyp1b1 in the neurovascular cells of the central nervous system including AC. We recently showed retinal AC constitutively express Cyp1b1, and global Cyp1b1-deficiency (Cyp1b1-/-) attenuates retinal ischemia-mediated neovascularization in vivo and the pro-angiogenic activity of retinal vascular cells in vitro. We also demonstrated that Cyp1b1 expression is a key regulator of retinal AC function. However, the underlying mechanisms involved need further investigation. Here we determined changes in the transcriptome profiles of Cyp1b1+/+ and Cyp1b1-/- retinal AC by RNA sequencing. We identified 585 differentially expressed genes, whose pathway enrichment analysis revealed the most significant pathways impacted in Cyp1b1-/- AC. These genes included those of axon guidance, extracellular matrix proteins and their receptors, cancer, cell adhesion molecules, TGF-β signaling, and the focal adhesion modulation. The expression of a selected set of differentially expressed genes was confirmed by RT-qPCR analysis. To our knowledge, this is the first report of RNAseq investigation of the retinal AC transcriptome and the molecular pathways impacted by Cyp1b1 expression. These results demonstrated an important role for Cyp1b1 expression in the regulation of various retinal AC functions, which are important in neurovascular development and integrity.