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Vascular endothelial damage due to ionizing radiation is the main pathological process of radiation injury and the main cause of damage to various organs in nuclear accidents. Ferroptosis plays an important role in ionizing radiation-induced cell death. We have previously reported that luteolin is highly resistant to ferroptosis. In the present study, body weight, microvessel count, H&E, and Masson staining results showed that luteolin rescued radial vascular injury in vivo. Cell Counting Kit 8 (CCK8), Giemsa staining clarified the anti-ferroptosis ability of luteolin with low toxicity. Malondialdehyde (MDA), superoxide dismutase (SOD), NADP+/NADPH, Fe2+ staining, dihydroethidium (DHE) and MitoTracker assays for ferroptosis-related metrics, we found that luteolin enhances human umbilical vein endothelial cells (HUVECs) antioxidant damage capacity. Drug affinity responsive target stability (DARTS), surface plasmon resonance (SPR), computer simulated docking and western blot showed that heat shock protein beta-1 (HSPB1) is one of the targets of luteolin action. Luteolin inhibits ferroptosis by promoting the protein expression of HSPB1/solute carrier family 7 member 11 (SLC7A11)/ glutathione peroxidase 4 (GPX4). In conclusion, we have preliminarily elucidated the antioxidant damage ferroptosis ability and the target of action of luteolin to provide a theoretical basis for the application of luteolin in radiation injury diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aims: Asprosin, a newly discovered hormone, is linked to insulin resistance. This study shows the roles of asprosin in vascular smooth muscle cell (VSMC) proliferation, migration, oxidative stress, and neointima formation of vascular injury. Methods: Mouse aortic VSMCs were cultured, and platelet-derived growth factor-BB (PDGF-BB) was used to induce oxidative stress, proliferation, and migration in VSMCs. Vascular injury was induced by repeatedly moving a guidewire in the lumen of the carotid artery in mice. Results: Asprosin overexpression promoted VSMC oxidative stress, proliferation, and migration, which were attenuated by toll-like receptor 4 (TLR4) knockdown, antioxidant (N-Acetylcysteine, NAC), NADPH oxidase 1 (NOX1) inhibitor ML171, or NOX2 inhibitor GSK2795039. Asprosin overexpression increased NOX1/2 expressions, whereas asprosin knockdown increased heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1) expressions. Asprosin inhibited nuclear factor E2-related factor 2 (Nrf2) nuclear translocation. Nrf2 activator sulforaphane increased HO-1 and NQO-1 expressions and prevented asprosin-induced NOX1/2 upregulation, oxidative stress, proliferation, and migration. Exogenous asprosin protein had similar roles to asprosin overexpression. PDGF-BB increased asprosin expressions. PDGF-BB-induced oxidative stress, proliferation, and migration were enhanced by Nrf2 inhibitor ML385 but attenuated by asprosin knockdown. Vascular injury increased asprosin expression. Local asprosin knockdown in the injured carotid artery promoted HO-1 and NQO-1 expressions but attenuated the NOX1 and NOX2 upregulation, oxidative stress, neointima formation, and vascular remodeling in mice. Innovation and Conclusion: Asprosin promotes oxidative stress, proliferation, and migration of VSMCs via TLR4-Nrf2-mediated redox imbalance. Inhibition of asprosin expression attenuates VSMC proliferation and migration, oxidative stress, and neointima formation in the injured artery. Asprosin might be a promising therapeutic target for vascular injury. Antioxid. Redox Signal. 41, 488-504.

Ostechondritis dissecans (OCD) is an orthopaedic disease characterized by formation of osteochondral defects in developing joints. Epiphyseal cartilage necrosis (osteochondrosis [OC]) caused by focal failure of vascular supply is the known precursor lesion of OCD, but it remains to be established how the severity of vascular failure drives lesion healing or progression. In the current study we have implemented a novel piglet model of induced osteochondrosis of the lateral trochlear ridge of the femur to determine the role that the extent of ischemia plays in the development and progression of OC/OCD lesions. Ten 4-week-old Yorkshire piglets underwent surgical interruption of the vascular supply to the entirety (n = 4 pigs) or the distal half (n = 6 pigs) of the lateral trochlear ridge of the femur. At 2, 6, and 12 weeks postoperatively, distal femora were evaluated by magnetic resonance imaging (MRI) to determine the fate of induced OC lesions. At 12 weeks, piglets were euthanized, and the surgical sites were examined histologically. After complete devascularization, lesion size increased between the 6- and 12-week MRI by an average of 24.8 mm2 (95% CI: [-2.2, 51.7]; p = 0.071). During the same period, lesion size decreased by an average of 7.6 mm2 (95% CI: [-24.5, 19.4]; p = 0.83) in piglets receiving partial devascularization. At 12 weeks, average ± SD lesion size was larger (p<0.001) in piglets undergoing complete (73.5 ± 17.6 mm2) vs. partial (16.5 ± 9.8 mm2) devascularization. Our study demonstrates how the degree of vascular interruption determines lesion size and likelihood of healing in a large animal model of trochlear OC., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tóth et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism., Methods and Results: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice ( P <0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice ( P <0.01 and P <0.05), but not in TLR3 -/- mice, by reinforcing neutrophil recruitment ( P <0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion ( P <0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation ( P <0.05). Finally, eRNA triggered plasma clot formation in vitro ( P <0.01)., Conclusions: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Acute vascular injury provokes an inflammatory response, resulting in neointimal hyperplasia (NIH) and downstream pathologies. The resolution of inflammation is an active process in which specialized proresolving lipid mediators (SPM) and their receptors play a central role. We sought to examine the acute phase response of SPM and their receptors in both circulating blood and the arterial wall in a rat angioplasty model. We found that the ratio of proresolving to pro-inflammatory lipid mediators (LM) in plasma decreased sharply 1 day after vascular injury, then increased slightly by day 7, while that in arteries remained depressed. Granulocyte expression of SPM receptors ALX/FPR2 and DRV2/GPR18, and a leukotriene B4 receptor BLT1 increased postinjury, while ERV1/ChemR23 expression was reduced early and then recovered by day 7. Importantly, we show unique arterial expression patterns of SPM receptors in the acute setting, with generally low levels through day 7 that contrasted sharply with that of the pro-inflammatory CCR2 receptor. Overall, these data document acute, time-dependent changes of LM biosynthesis and SPM receptor expression in plasma, leukocytes, and artery walls following acute vascular injury. A biochemical imbalance between inflammation and resolution LM pathways appears persistent 7 days after angioplasty in this model. These findings may help guide therapeutic approaches to accelerate vascular healing and improve the outcomes of vascular interventions for patients with advanced atherosclerosis., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: Intimal hyperplasia is the response to endothelial injury. Platelet-derived growth factor is released early and favors the formation of intimal hyperplasia. Although multiple treatments, from open surgery to endovascular techniques, have been used they remain controversial. There is currently interest in developing pharmacological strategies to address this pathology. Local vascular inflammation induced by vessel barotrauma generates intimal hyperplasia due to mechanical stress over the venous endothelium. Cilostazol is a selective phosphodiesterase type 3 (PDE3) selective inhibitor with a regulatory effect over intimal hyperplasia. The objective was to investigate cilostazol's role in inhibiting smooth muscle cell proliferation due to changes in the expression and release of PDGF-BB isoform and the effect on developing IH using an experimental model of vascular barotrauma (balloon-induced injury model)., Methods: We included 12 New Zealand rabbits. The balloon-induced injury model (BIIM) and experimental group cilostazol (20 mg/kg/day) included 6 rabbits each. Contralateral veins from 6 rabbits used in BIIM model has been taken as control group. We measured and compared the expression of PDGF-BB and the development of IH. A pathologist board chooses a PDGFRα antibody to localized its expression by immunohistochemistry analysis. Subsequently, using an automated immunohistochemical staining machine, the PDGFR expression was evaluated using a Zeiss Primo Star 4 light microscope., Results: The measurement obtained in the intimal layer was: 126.12 μm2 in the CG, 232 μm2 in the BIIM group, and 178 μm2 in the EG. A statistically significant difference was observed. Baseline serum concentrations of PDGF-BB in the BIIM group were 0.22 pg/mL. At 12 h 0.42 pg/mL, and 0.17 pg/mL at seven days. In the experimental group, the basal levels were 0.33 pg/mL. With the use of cilostazol, a lower peak was obtained at 12 h (0.08 pg/mL). This difference was statistically significant., Conclusions: Cilostazol induced a significant reduction of IH caused by barotrauma in the venous endothelium, which correlates with decrease in the PDGF-BB in serum. This could be attributed to the pharmacologic effect on PDGFR expression., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Following the placement of endovascular implants, perivascular adipose tissue (PVAT) becomes an early sensor of vascular injury to which it responds by undergoing phenotypic changes characterized by reduction in the secretion of adipocyte-derived relaxing factors and a shift to a proinflammatory and pro-contractile state. Thus, activated PVAT loses its anti-inflammatory function, secretes proinflammatory cytokines and chemokines, and generates reactive oxygen species, which are accompanied by differentiation of fibroblasts into myofibroblasts and proliferation of smooth muscle cells. These subsequently migrate into the intima, leading to intimal growth. In addition, periadventitial vasa vasorum undergoes neovascularization and functions as a portal for extravasation of inflammatory infiltrates and mobilization of PVAT resident stem/progenitor cells into the intima. This review focuses on the response of PVAT to endovascular intervention-induced injury and discusses potential therapeutic targets to suppress the PVAT-initiated pathways that mediate the formation of neointima., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Ischemic vascular diseases are on the rise globally, including ischemic heart diseases, ischemic cerebrovascular diseases, and ischemic peripheral arterial diseases, posing a significant threat to life. Copper is an essential element in various biological processes, copper deficiency can reduce blood vessel elasticity and increase platelet aggregation, thereby increasing the risk of ischemic vascular disease; however, excess copper ions can lead to cytotoxicity, trigger cell death, and ultimately result in vascular injury through several signaling pathways. Herein, we review the role of cuproptosis and copper deficiency implicated in ischemic injury and repair including myocardial, cerebral, and limb ischemia. We conclude with a perspective on the therapeutic opportunities and future challenges of copper biology in understanding the pathogenesis of ischemic vascular disease states., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Central venous catheters (CVCs) are often trimmed during heart transplantation and pediatric cardiac surgery. However, the risk of endothelial injury caused by the cut tip of the CVC has not been evaluated. We hypothesized that there is no difference in the degree of endothelial injury associated with trimmed CVCs versus standard untrimmed CVCs., Methods: In four adult male sheep, the left external jugular vein was exposed in three segments, one designated for an untouched control group, one for the trimmed CVC group, and one for the untrimmed CVC group. Trimmed and untrimmed CVC tips were rotated circumferentially within their respective segments to abrade the lumen of the vein. The vein samples were explanted, and two representative sections from each sample were analyzed using hematoxylin and eosin (H&E) staining, as well as with immunohistochemistry against CD31, von Willebrand factor (vWF), endothelial nitric oxide synthase (eNOS), and caveolin. Higher immunohistochemical stain distributions and intensities are associated with normal health and function of the venous endothelium. Data are presented as counts with percentages or as means with standard error., Results: H&E staining revealed no evidence of endothelial injury in 6/8 (75%) samples from the untouched control group, and no injury in 4/8 (50%) samples from both the trimmed and untrimmed CVC groups ( p  = 0.504). In all remaining samples from each group, only mild endothelial injury was observed. Immunohistochemical analysis comparing trimmed CVCs versus untrimmed CVCs revealed no difference in the percentage of endothelial cells staining positive for CD31 (57.5% ± 7.2% vs 55.0% ± 9.2%, p  = 0.982), vWF (73.8% ± 8.0% vs 62.5% ± 9.6%, p  = 0.579), eNOS (66.3% ± 4.2% vs 63.8% ± 7.5%, p  = 0.962), and caveolin (53.8% ± 5.0% vs 51.3% ± 4.4%, p  = 0.922). There were no significant differences between the groups in the distributions of stain intensity for CD31, vWF, eNOS, and caveolin., Conclusion: Trimmed CVCs do not increase endothelial injury compared to standard untrimmed CVCs., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The RNA-binding zinc finger protein 36 (ZFP36) family participates in numerous physiological processes including transition and differentiation through post-transcriptional regulation. ZFP36L1 is a member of the ZFP36 family. This study aimed to evaluate the role of ZFP36L1 in restenosis. We found that the expression of ZFP36L1 was inhibited in VSMC-phenotypic transformation induced by TGF-β, PDGF-BB, and FBS and also in the rat carotid injury model. In addition, we found that the overexpression of ZFP36L1 inhibited the proliferation and migration of VSMCs and promoted the expression of VSMC contractile genes; whereas ZFP36L1 interference promoted the proliferation and migration of VSMCs and suppressed the expression of contractile genes. Furthermore, the RNA binding protein immunoprecipitation and double luciferase reporter gene experiments shows that ZFP36L1 regulates the phenotypic transformation of VSMCs through the posttranscriptional regulation of KLF16. Finally, our research results in the rat carotid balloon injury animal model further confirmed that ZFP36L1 regulates the phenotypic transformation of VSMCs through the posttranscriptional regulation of KLF16 and further plays a role in vascular injury and restenosis in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024. Published by Elsevier Ltd.)

Endothelial repair is essential for restoring tissue fluid homeostasis following lung injury. R-spondin3 (RSPO3), a secreted protein mainly produced by endothelial cells (ECs), has shown its protective effect on endothelium. However, the specific mechanisms remain unknown. To explore whether and how RSPO3 regulates endothelial regeneration after inflammatory vascular injury, the role of RSPO3 in sepsis-induced pulmonary endothelial injury was investigated in EC-specific RSPO3 knockdown, inducible EC-specific RSPO3 deletion mice, EC-specific RSPO3 overexpression mice, systemic RSPO3-administration mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in plasma from septic patients. Here we show that plasma RSPO3 levels are decreased in septic patients and correlated with endothelial injury markers and PaO 2 /FiO 2 index. Both pulmonary EC-specific knockdown of RSPO3 and inducible EC-specific RSPO3 deletion inhibit pulmonary ECs proliferation and exacerbate ECs injury, whereas intra-pulmonary EC-specific RSPO3 overexpression promotes endothelial recovery and attenuates ECs injury during endotoxemia. We show that RSPO3 mediates pulmonary endothelial regeneration by a LGR4-dependent manner. Except for β-catenin, integrin-linked kinase (ILK)/Akt is also identified as a novel downstream effector of RSPO3/LGR4 signaling. These results conclude that EC-derived RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of β-catenin and ILK signaling pathways after inflammatory vascular injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation., Methods: Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function., Results: Phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, and extracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression., Conclusion: These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanoplastics pose several health hazards, especially vascular toxicity. Transfer RNA-derived small RNAs (tsRNAs) are novel noncoding RNAs associated with different pathological processes. However, their biological roles and mechanisms in aberrant vascular smooth muscle cell (VSMC) plasticity and vascular injury are unclear. This study investigated the potent effects of tsRNAs on vascular injury induced by short- and long-term exposure to polystyrene nanoplastics (PS-NPs). Mice were exposed to PS-NPs (100 nm) at different doses (10-100 μg/mL) for 30 or 180 days. High-throughput sequencing was used to analyze tsRNA expression patterns in arterial tissues obtained from an in vivo model. Additionally, quantitative real-time polymerase chain reaction, fluorescent in situ hybridization assays, and dual-luciferase reporter assays were performed to measure the expression and impact of tiRNA-Glu-CTC on VSMCs exposed to PS-NPs. Short-term (≥50 μg/mL, moderate concentration) and long-term (≥10 μg/mL, low concentration) PS-NP exposure induced vascular injury in vivo. Cellular experiments showed that the moderate concentration of PS-NPs induced VSMC phenotypic switching, whereas a high concentration of PS-NPs (100 μg/mL) promoted VSMC apoptosis. PS-NP induced severe mitochondrial damage in VSMCs, including overexpression of reactive oxygen species, accumulation of mutated mtDNA, and dysregulation of genes related to mitochondrial synthesis and division. Compared with the control group, 13 upregulated and 12 downregulated tRNA-derived stress-induced RNAs (tiRNAs) were observed in the long-term PS-NP (50 μg/mL) exposure group. Bioinformatics analysis indicated that differentially expressed tiRNAs targeted genes that were involved in vascular smooth muscle contraction and calcium signaling pathways. Interestingly, tiRNA-Glu-CTC was overexpressed in vivo and in vitro following PS-NP exposure. Functionally, the tiRNA-Glu-CTC inhibitor mitigated VSMC phenotypic switching and mitochondrial damage induced by PS-NP exposure, whereas tiRNA-Glu-CTC mimics had the opposite effect. Mechanistically, tiRNA-Glu-CTC mimics induced VSMC phenotypic switching by downregulating Cacna1f expression. PS-NP exposure promoted VSMC phenotypic switching and vascular injury by targeting the tiRNA-Glu-CTC/Cacna1f axis., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Vascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease., Methods: Wire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype., Results: Phenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation., Conclusions: The present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis., (© 2024. The Author(s).)

Pathological vascular remodeling is a hallmark of various vascular diseases. Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction, which leads to pathological vascular remodeling. Potassium dehydroandrographolide succinate (PDA), a derivative of andrographolide, has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections. This study investigates the potential of PDA in regulating pathological vascular remodeling. The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice. Experimental approaches, including rat aortic primary smooth muscle cell culture, flow cytometry, bromodeoxyuridine (BrdU) incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay, were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells (SMCs). Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions. The results revealed that PDA exacerbates vascular injury-induced pathological remodeling, as evidenced by enhanced neointima formation. PDA treatment significantly increased the proliferation and migration of SMCs. Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88 (MyD88) expression in SMCs and interacted with T-cadherin (CDH13). This interaction augmented proliferation, migration, and extracellular matrix deposition, culminating in pathological vascular remodeling. Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling, mediated through the MyD88/CDH13 signaling pathway., (Copyright © 2024 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Introduction: This study aimed to determine whether bone morphogenetic protein-4 (BMP-4), which increases in response to intimal hyperplasia, promotes phenotype transition in vascular smooth muscle cells (VSMCs)., Methods: Balloon injury was used to induce intimal hyperplasia in rats. Hematoxylin-eosin staining was used to detect the alteration of vascular structure. Serum levels of BMP-4 and lactate were detected by ELISA. Human aortic smooth muscle cells (HA-SMCs) were cultured. Protein and mRNA expression levels were detected through Western blot and real-time PCR. Cell migration was measured by transwell assay., Results: Our data showed that serum concentration of BMP-4 was upregulated after balloon injury. Treatment with BMP-4 inhibitor DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo(1,5-a)pyrimidin-3-yl)quinoline) suppressed the abnormal expression of BMP-4 and inhibited the intimal hyperplasia induced by balloon injury. Compared to BMP-4-negative medium, BMP-4-positive medium was associated with higher synthetic VSMC marker expression levels and lower in contractile gene markers in cultured HA-SMCs. Transfection of monocarboxylic acid transporters-4 (MCT-4) siRNA inhibited the excretion of lactate induced by BMP-4., Conclusion: Our analyses provided evidence that BMP-4 and its regulator Smad-4 are key regulators in MCT-4-mediated lactate excretion. This indicates that BMP-4 stimulates the phenotypic transition of VSMCs via SMAD-4/MCT-4 signaling pathway., (© 2023 S. Karger AG, Basel.)

Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. To develop a vascular adventitial drug delivery system to treat intimal hyperplasia post vascular injuries, we loaded miR-145-5p-agomir (miR-145) into an injectable and in-situ self-assembling RAD peptide hydrogel. In vitro data showed that the miR-145 could be well incorporated into the RAD peptide hydrogels and released in a slow and controlled manner. The released miR-145 could transfect SMCs successfully, and the transfected SMCs exhibited a reduced migration capacity and higher expressions of SMC contractile biomarkers as compared to the non-transfected SMCs. In vivo data showed that the retention of the miR-145 was greatly elongated by the RAD peptide hydrogels. In addition, the application of the miR-145-loaded RAD peptide hydrogels surrounding injured arteries decreased the proliferative SMCs, promoted the regeneration of endothelium, reduced the macrophage infiltration, inhibited the neointimal formation and prevented adverse ECM remodeling via downregulation of KLF4 expression. The RAD peptide hydrogels loaded with miR-145 can successfully inhibit intimal hyperplasia after vascular injuries and thus hold great potential as an innovative extravascular drug delivery approach to treat vascular diseases. STATEMENT OF SIGNIFICANCE: Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. Our work here demonstrates that the RAD peptide hydrogels loaded with miR-145-5p-agomir (miR-145) can successfully reverse intimal hyperplasia after vascular injuries and thus hold great potential as an innovative vascular adventitial drug delivery approach to treat vascular diseases. Our work proposes a possible paradigm shift from endovascular drug delivery to extravascular drug delivery for vascular disorder treatment., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: Diabetes pathology relies on exosomes (Exos). This study investigated how peripheral blood Exo-containing microRNAs (miRNAs) cause vascular injury in type 2 diabetes (T2D)., Methods: We removed DEmiRNA from T2D chip data from the GEO database. We isolated Exo from 15 peripheral blood samples from T2D patients and 15 healthy controls and measured Exo DEmiRNA levels. We employed the intersection of Geneards and mirWALK database queries to find T2D peripheral blood mRNA-related chip target genes. Next, we created a STRING database candidate target gene interaction network map. Next, we performed GO and KEGG enrichment analysis on T2D-related potential target genes using the ClusterProfiler R package. Finally, we selected T2D vascular damage core genes and signaling pathways using GSEA and PPI analysis. Finally, we used HEK293 cells for luciferase assays, co-cultured T2D peripheral blood-derived Exo with HVSMC, and detected HVSMC movement alterations., Results: We found 12 T2D-related DEmiRNAs in GEO. T2D patient-derived peripheral blood Exo exhibited significantly up-regulated miR-135a-3p by qRT-PCR. Next, we projected miR-135a-3p's downstream target mRNA and screened 715 DEmRNAs to create a regulatory network diagram. DEmRNAs regulated biological enzyme activity and vascular endothelial cells according to GO function and KEGG pathway analysis. ErbB signaling pathway differences stood out. PPI network study demonstrated that DEmRNA ATM genes regulate the ErbB signaling pathway. The luciferase experiment validated miR-135a-3p and ATM target-binding. Co-culture of T2D patient-derived peripheral blood Exo with HVSMC cells increases HVSMC migration, ErbB2, Bcl-2, and VEGF production, and decreases BAX and ATM. However, miR-135a-3p can reverse the production of the aforesaid functional proteins and impair HVSMC cell movement., Conclusion: T2D patient-derived peripheral blood Exo carrying miR-135a-3p enter HVSMC, possibly targeting and inhibiting ATM, activating the ErbB signaling pathway, promoting abnormal HVSMC proliferation and migration, and aggravating vascular damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xie, Li, Wang, Fu and Cai.)

Background: Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis., Methods: We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition., Results: During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-β/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation., Conclusions: Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

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

Abstract: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) cause neointimal hyperplasia after percutaneous vascular interventions. Nuclear receptor subfamily 1 group D member 1 (NR1D1), a crucial member of circadian clock, is involved in the regulation of atherosclerosis and cellular proliferation. However, whether NR1D1 affects vascular neointimal hyperplasia remains unclear. In this study, we found that activating NR1D1 reduced injury-induced vascular neointimal hyperplasia. Overexpression of NR1D1 reduced the number of Ki-67-positive VSMCs and migrated VSMCs after platelet-derived growth factor (PDGF)-BB treatment. Mechanistically, NR1D1 suppressed the phosphorylation of AKT and 2 main effectors of the mammalian target of rapamycin complex 1 (mTORC1), S6, and 4EBP1 in PDGF-BB-challenged VSMCs. Re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (si Tsc1 ) and re-activation of AKT by SC-79 abolished NR1D1-mediated inhibitory effects on proliferation and migration of VSMCs. Moreover, decreased mTORC1 activity induced by NR1D1 was also reversed by SC-79. Simultaneously, Tsc1 knockdown abolished the vascular protective effects of NR1D1 in vivo. In conclusion, NR1D1 reduces vascular neointimal hyperplasia by suppressing proliferation and migration of VSMCs in an AKT/mTORC1-dependent manner., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Mechanotransduction is the ability of cells to "feel" or sense their mechanical microenvironment and integrate and convert these physical stimuli into adaptive biochemical cellular responses. This phenomenon is vital for the physiology of numerous nucleated cell types to affect their various cellular processes. As the main drivers of hemostasis and clot retraction, platelets also possess this ability to sense the dynamic mechanical microenvironments of circulation and convert those signals into biological responses integral to clot formation. Like other cell types, platelets leverage their "hands" or receptors/integrins to mechanotransduce important signals in responding to vascular injury to achieve hemostasis. The clinical relevance of cellular mechanics and mechanotransduction is imperative as pathologic alterations or aberrant mechanotransduction in platelets has been shown to lead to bleeding and thrombosis. As such, the aim of this review is to provide an overview of the most recent research related to platelet mechanotransduction, from platelet generation to platelet activation, within the hemodynamic environment and clot contraction at the site of vascular injury, thereby covering the entire "life cycle" of platelets. Additionally, we describe the key mechanoreceptors in platelets and discuss the new biophysical techniques that have enabled the field to understand how platelets sense and respond to their mechanical microenvironment via those receptors. Finally, the clinical significance and importance of continued exploration of platelet mechanotransduction have been discussed as the key to better understanding of both thrombotic and bleeding disorders lies in a more complete mechanistic understanding of platelet function by way of mechanotransduction., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Purpose: The association between the extent of the wire and device bias as assessed by optical coherence tomography (OCT) in the healthy portion of the vessel and the risk of coronary artery injury after orbital atherectomy (OA) has not been fully elucidated. Thus, purpose of this study is to investigate the association between pre-OA OCT findings and post-OA coronary artery injury by OCT., Methods: We enrolled 148 de novo lesions having calcified lesion required OA (max Ca angle > 90°) in 135 patients who underwent both pre- and post-OA OCT. In pre-OA OCT, OCT catheter contact angle and the presence or absences of guide-wire (GW) contact with the normal vessel intima were assessed. Also, in post-OA OCT, we assessed there was post-OA coronary artery injury (OA injury), defined as disappearance of both of intima and medial wall of normal vessel, or not., Results: OA injury was found in 19 lesions (13%). Pre-PCI OCT catheter contact angle with the normal coronary artery was significantly larger (median 137°; inter quartile range [IQR] 113-169 vs. median 0°; IQR 0-0, P < 0.001) and more GW contact with the normal vessel was found (63% vs. 8%, P < 0.001). Pre-PCI OCT catheter contact angle > 92° and GW contact with the normal vessel intima were associated with post-OA vascular injury (Both: 92% (11/12), Either: 32% (8/25), Neither: 0% (0/111), P < 0.001)., Conclusion: Pre-PCI OCT findings, such as catheter contact angle > 92° and guide-wire contact to the normal coronary artery, were associated with post-OA coronary artery injury., (© 2023. The Author(s).)

Cardiovascular disease (CVD) is the leading cause of death worldwide and is primarily associated with obesity, visceral adiposity, and unhealthy perivascular adipose tissue (PVAT). The inflammatory polarization of immune cells residing in adipose tissue and abnormal levels of adipose-related cytokines are crucial factors contributing to the pathogenesis of metabolic disorders. We reviewed the most relevant papers in the English literature regarding PVAT and obesity-related inflammation and CVD, aiming to explore potential therapeutic targets for metabolic alterations related to CV health. Such an understanding will help determine the pathogenetic relationship between obesity and vascular injury in efforts to ameliorate obesity-related inflammatory responses. In the context of obesity, dysregulation of adipose tissue immune function, which consists of immune cells and adipose-derived cytokines, plays a crucial role in vascular injury and endothelial dysfunction, especially the PVAT. Metabolic changes between typical VAT and PVAT in obese conditions would be beneficial in improving the risk of obesity-induced endothelial dysfunction and CVDs., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

Background: Restenosis after percutaneous coronary intervention (PCI) limits therapeutic revascularization. Neuropeptide Y (NPY), co-stored and co-released with the sympathetic nervous system, is involved in this process, but its exact role and underlying mechanisms remain to be fully understood. This study aimed to investigate the role of NPY in neointima formation after vascular injury., Methods: Using the left carotid arteries of wild-type (WT, NPY-intact) and NPY-deficient (NPY -/- ) mice, ferric chloride-mediated carotid artery injury induced neointima formation. Three weeks after injury, the left injured carotid artery and contralateral uninjured carotid artery were collected for histological analysis and immunohistochemical staining. RT-qPCR was used to detect the mRNA expression of several key inflammatory markers and cell adhesion molecules in vascular samples. Raw264.7 cells were treated with NPY, lipopolysaccharide (LPS), and lipopolysaccharide-free, respectively, and RT-qPCR was used to detect the expression of these inflammatory mediators., Results: Compared with WT mice, NPY -/- mice had significantly reduced neointimal formation three weeks after injury. Mechanistically, immunohistochemical analysis showed there were fewer macrophages and more vascular smooth muscle cells in the neointima of NPY -/- mice. Moreover, the mRNA expression of key inflammatory markers such as interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), and intercellular adhesion molecule-1 (ICAM-1) was significantly lower in the injured carotid arteries of NPY -/- mice, compared to that in the injured carotid arteries of WT mice. In RAW264.7 macrophages, NPY significantly promoted TGF-β1 mRNA expression under unactivated but not LPS-stimulated condition., Conclusions: Deletion of NPY attenuated neointima formation after artery injury, at least partly, through reducing the local inflammatory response, suggesting that NPY pathway may provide new insights into the mechanism of restenosis., (© 2023. The Author(s).)

Doxorubicin (DOX) is a widely used powerful anthracycline for treatment of many varieties of malignancies; however its cumulative and dose-dependent cardio-toxicity has been limited its clinical use. In the current study, in vivo and in vitro (neonatal rat's cardiomyocytes) experiments were conducted to identify the impact of nifuroxazide (NIFU) on DOX-induced cardiomyopathy, vascular injury, and hemato-toxcity and plot the underlying regulatory mechanisms. Cardiovascular injury was induced in vivo by I.P. injection of an overall dose of DOX (21 mg/kg) administered (3.5 mg/kg) twice weekly for 21 days. NIFU (10 and 30 mg/kg) was administered orally once daily for 21 days, 1 week after DOX injection initiation. In vivo experiments confirmed NIFU to restore blood cells counts and hemoglobin concentration. Moreover, NIFU normalized the myocardial functional status as confirmed by ECG examination and myocardial injury markers; CK-MB, LDH, and AST. NIFU restored the balance between TAC and both of ROS and MDA and down-regulated the protein expression of TLR4, NF-kB, TXNIP, NLR-family pyrin domain containing 3 (NLRP3), caspase-1, IL-1β, and GSDMD-N terminal, with inhibition of the up-stream of NLRP3 and the down-stream DOX-induced pyroptosis. The in vitro assay confirmed well preserved cardiomyocytes' architecture, amelioration of NLRP3/IL-1 β-mediated cell pyroptosis, enhanced cell viability, and improved spontaneous beating. Moreover, NIFU normalized the disturbed aortic oxidant-antioxidant balance; enhanced eNOS- mediated endothelial relaxation, and down regulated IL-1β expression. Thus, NIFU may be proposed to serve as a cardioprotective agent to attenuate DOX-induced cardio-toxicity and vascular injury., 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 © 2022. Published by Elsevier Inc.)

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-β (PI3Kβ) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kβ inhibitor-treated, or endothelial-selective PI3Kβ knockout (ECβKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kβ-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECβKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECβKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kβ inhibition or RNA interference. Selective PI3Kβ inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kβ as a therapeutic target to reduce vascular inflammation and injury., (Copyright © 2022 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: This study examined neuropathological findings of patients who died following hospitalization in an intensive care unit with SARS-CoV-2., Methods: Data originate from 20 decedents who underwent brain autopsy followed by ex-vivo imaging and dissection. Systematic neuropathologic examinations were performed to assess histopathologic changes including cerebrovascular disease and tissue injury, neurodegenerative diseases, and inflammatory response. Cerebrospinal fluid (CSF) and fixed tissues were evaluated for the presence of viral RNA and protein., Results: The mean age-at-death was 66.2 years (range: 26-97 years) and 14 were male. The patient's medical history included cardiovascular risk factors or diseases (n = 11, 55%) and dementia (n = 5, 25%). Brain examination revealed a range of acute and chronic pathologies. Acute vascular pathologic changes were common in 16 (80%) subjects and included infarctions (n = 11, 55%) followed by acute hypoxic/ischemic injury (n = 9, 45%) and hemorrhages (n = 7, 35%). These acute pathologic changes were identified in both younger and older groups and those with and without vascular risk factors or diseases. Moderate-to-severe microglial activation were noted in 16 (80%) brains, while moderate-to-severe T lymphocyte accumulation was present in 5 (25%) brains. Encephalitis-like changes included lymphocytic cuffing (n = 6, 30%) and neuronophagia or microglial nodule (most prominent in the brainstem, n = 6, 30%) were also observed. A single brain showed vasculitis-like changes and one other exhibited foci of necrosis with ball-ring hemorrhages reminiscent of acute hemorrhagic leukoencephalopathy changes. Chronic pathologies were identified in only older decedents: 7 brains exhibited neurodegenerative diseases and 8 brains showed vascular disease pathologies. CSF and brain samples did not show evidence of viral RNA or protein., Conclusions: Acute tissue injuries and microglial activation were the most common abnormalities in COVID-19 brains. Focal evidence of encephalitis-like changes was noted despite the lack of detectable virus. The majority of older subjects showed age-related brain pathologies even in the absence of known neurologic disease. Findings of this study suggest that acute brain injury superimposed on common pre-existing brain disease may put older subjects at higher risk of post-COVID neurologic sequelae., (© 2022. The Author(s).)

Introduction: Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes., Methods: We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice., Results: We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo., Conclusion: We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Dantrolene is a ryanodine receptor blocker that is used clinically for treatment of malignant hyperthermia. This study was conducted using murine aortic vascular smooth muscle cells (MOVAS) and a mouse arterial injury model to investigate the inhibitory effect of dantrolene on smooth muscle cell proliferation and migration. We investigated whether dantrolene suppressed platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and migration in vitro. The effect of dantrolene on smooth muscle phenotype was evaluated using immunostaining. In addition, smooth muscle cell proliferation and phenotype switching were tested by applying dantrolene around blood vessels using a mouse arterial injury model. Dantrolene inhibited PDGF-induced cell proliferation and migration of MOVAS. Dantrolene also inhibited the switch from contractile to synthetic phenotype both in vitro and in vivo. Dantrolene is effective at inhibiting vascular smooth muscle cell proliferation, migration, and neointimal formation following arterial injury in mice., Competing Interests: Declaration of competing interest All authors have no conflict of interest in this study., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background:  Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury., Methods:  Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ER T2 recombinase under the Myh11 promoter with PTP1B flox/flox mice and subjected to FeCl 3 carotid artery injury., Results:  Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA + and PDGFRβ + myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11 -Cre reporter cells in the remodeling adventitia, and SCA1 + CD45 - vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor β (TGFβ) signaling components or enzymes involved in extracellular matrix remodeling and TGFβ liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFRβ and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion., Conclusion:  Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Although inflammation plays critical roles in the development of atherosclerosis, its regulatory mechanisms remain incompletely understood. Perivascular adipose tissue (PVAT) has been reported to undergo inflammatory changes in response to vascular injury. Here, we show that vascular injury induces the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulate in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by genetically silencing PRDM16, a key regulator to beiging, exacerbates inflammation and vascular remodeling following injury. Conversely, activation of PVAT beiging attenuates inflammation and pathological vascular remodeling. Single-cell RNA sequencing reveals that beige adipocytes abundantly express neuregulin 4 (Nrg4) which critically regulate alternative macrophage activation. Importantly, significant beiging is observed in the diseased aortic PVAT in patients with acute aortic dissection. Taken together, vascular injury induces the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitates alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrates the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis., (© 2022. The Author(s).)

The purpose of our study was to investigate if vascular injury in immature epiphyses affects cartilage repair outcomes of matrix-associated stem cell implants (MASI). Porcine bone marrow mesenchymal stromal stem cells (BMSCs) suspended in a fibrin glue scaffold were implanted into 24 full-thickness cartilage defects (5 mm ø) of the bilateral distal femur of six Göttingen minipigs (n = 12 defects in 6 knee joints of 3 immature pigs; age 3.5-4 months; n = 12 defects in 6 knee joints of 3 mature control pigs; age, 21-28 months). All pigs underwent magnetic resonance imaging (MRI) at 2, 4, 12 (n = 24 defects), and 24 weeks (n = 12 defects). After the last imaging study, pigs were sacrificed, joints explanted and evaluated with VEGF, H&E, van Gieson, Mallory, and Safranin O stains. Results of mature and immature cartilage groups were compared using the Wilcoxon signed-rank test. Quantitative scores for subchondral edema at 2 weeks were correlated with quantitative scores for cartilage repair (MOCART score and ICRS score) at 12 weeks as well as Pineda scores at end of the study, using linear regression analysis. On serial MRIs, mature joints demonstrated progressive healing of cartilage defects while immature joints demonstrated incomplete healing and damage of the subchondral bone. The MOCART score at 12 weeks was significantly higher for mature joints (79.583 ± 7.216) compared to immature joints (30.416 ± 10.543, p = 0.002). Immature cartilage demonstrated abundant microvessels while mature cartilage did not contain microvessels. Accordingly, cartilage defects in immature joints showed a significantly higher number of disrupted microvessels, subchondral edema, and angiogenesis compared to mature cartilage. Quantitative scores for subchondral edema at 2 weeks were negatively correlated with MOCART scores (r =  - 0.861) and ICRS scores (r =  - 0.901) at 12 weeks and positively correlated with Pineda scores at the end of the study (r = 0.782). Injury of epiphyseal blood vessels in immature joints leads to subchondral bone defects and limits cartilage repair after MASI., (© 2022. The Author(s).)

Background: Vascular damage, autoimmune abnormalities, and fibrosis are the three pathological features of systemic sclerosis (SSc).However, pulmonary vascular damage is the main factor affecting the progression and prognosis of SSc. The main purpose of this study was to explore the molecular mechanism of Wenyang Huazhuo Tongluo Formula in alleviating pulmonary vascular injury in bleomycin-induced SSc mouse model., Methods: Masson staining and H&E staining were used to analyze the degree of pulmonary vascular fibrosis and the infiltration of leukocyte cells in lung tissue ofbleomycin-induced SSc mouse models treated with saline (BLM group), Wenyang Huazhuo Tongluo Formula (WYHZTL group) and HIF-1α inhibitor KC7F2 (KC7F2 group). Blood vessel exudation was determined by analyzing the cell number and albumin concentration in bronchoalveolar lavage fluid using a cell counter and ELISA assay, respectively. The degree of vascular injury was assessed by measuring the expression levels of vWF, E-selectin, ICAM-1, VCAM-1, VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells using ELISA and immunofluorescence staining. Finally, the effect of Wenyang Huazhuo Tongluo Formula on the expression of HIF-1α was detected using immunofluorescence staining., Results: Wenyang Huazhuo Tongluo Formula and KC7F2 significantly inhibited bleomycin-induced pulmonary vascular fibrosis and the level of perivascular inflammatory cell infiltration. The number of cells and the concentration of albumin were significantly reduced in the bronchoalveolar lavage fluid of the WYHZTL group and KC7F2 group compared with the BLM group. In addition, treatment with Wenyang Huazhuo Tongluo Formula and KC7F2 significantly downregulated the expression levels of vWF, E-selectin, ICAM-1, VCAM-1 and HIF-1α, but upregulated the expression of VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells, compared with treatment with saline., Conclusions: This study reveals that Wenyang Huazhuo Tongluo Formula plays a new role in the treatment of SSc by alleviating pulmonary vascular damage. Furthermore, we found that Wenyang Huazhuo Tongluo Formula alleviates pulmonary vascular injury and inhibits HIF-1α expression., (© 2022. The Author(s).)

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

Abnormal proliferation and migration of vascular smooth muscle cell (VSMC) is a hallmark of vascular neointima hyperplasia. Perilipin 5 (Plin5), a regulator of lipid metabolism, is also confirmed to be involved in vascular disorders, such as microvascular endothelial dysfunction and atherosclerosis. To investigate the regulation and function of plin5 in the phenotypic alteration of VSMC, -an animal model of vascular intima hyperplasia was established in C57BL/6 J and Plin5 knockdown (Plin5 ± ) mice by wire injure. Immunohistochemical staining was used to analyze neointima hyperplasia in artery. Ki-67, dihydroethidium immunofluorescence staining and wound healing assay were used to measure proliferation, reactive oxygen species (ROS) generation and migration of VSMC, respectively. Plin5 was downregulated in artery subjected to vascular injury and in VSMC subjected to platelet-derived growth factor (PDGF)-BB. Plin5 knockdown led to accelerated neointima hyperplasia, excessive proliferation and migration of VSMC after injury. In vitro, we observed increased ROS content in VSMC isolated from Plin5 ± mice. Antioxidative N-acetylcysteine (NAC) inhibited VSMC proliferation and migration induced by PDGF-BB or plin5 knockdown. More importantly, plin5-peroxlsome proliferator-activated receptor-γ coactivator (PGC)-1α interaction was also attenuated in VSMC after knockdown of plin5. Overexpression of PGC-1α suppressed PDGF-BB-induced ROS generation, proliferation, and migration in VSMC isolated from Plin5 ± mice. These data suggest that plin5 serves as a potent regulator of VSMC proliferation, migration, and neointima hyperplasia by interacting with PGC-1α and affecting ROS generation.

IMP3, an RNA-binding protein (RBP) that participates in the process of post-transcriptional modifications of mRNA transcripts, is capable of altering cellular functions, and in some cases, be involved in specific disease progression. We aimed to investigate whether IMP3 has the ability to regulate the functional properties of endothelial cells and re-endothelialization in response to arterial injury. Wire injury was introduced to the right carotid arteries of wildtype C57/BL6 mice. As a result, IMPs' expressions were up-regulated in the induced arterial lesions, and IMP3 was the most up-regulated RNA among other IMPs. We overexpressed IMP3 before the wire-injured surgery using adeno-associated virus AAV2-IMP3. In vivo studies confirmed that IMP3 overexpression accelerated the progress of re-endothelialization after arterial injury. In vitro, endothelial cells were transfected with either ad-IMP3 or Si-IMP3, cell functional studies showed that IMP3 could promote endothelial cell proliferation and migration, while reducing apoptosis. Mechanistic studies also revealed that IMP3 could enhance VEGF mRNA stability and therefore up-regulate activities of VEGF/PI3K/Akt signalling pathway. Our data indicated that IMP3 promotes re-endothelialization after arterial injury and regulates endothelial cell proliferation, migration and apoptosis via increasing stability of VEGF mRNA and activation of VEGF/PI3K/Akt signalling pathway., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background:  Smooth muscle cells (SMCs) are the main driver of neointima formation and restenosis following vascular injury. In animal models, endothelial progenitor cells (EPCs) accelerate endothelial regeneration and reduce neointima formation after arterial injury; however, EPC-capture stents do not reduce target vessel failure compared with conventional stents. Here we examined the influence of EPCs on features of SMCs pivotal for their impact on injury-induced neointima formation including proliferation, migration, and phenotype switch., Methods and Results:  EPCs, their conditioned medium, and EPC-derived microparticles induced proliferation of SMCs while limiting their apoptosis. In transwell membrane experiments and scratch assays, EPCs stimulated migration of SMCs and accelerated their recovery from scratch-induced injury. Treatment of SMCs with an EPC-derived conditioned medium or microparticles triggered transformation of SMCs toward a synthetic phenotype. However, co-cultivation of EPCs and SMCs enabling direct cell-cell contacts preserved their original phenotype and protected from the transformative effect of SMC cholesterol loading. Adhesion of EPCs to SMCs was stimulated by SMC injury and reduced by blocking CXCR2 and CCR5. Interaction of EPCs with SMCs modulated their secretory products and synergistically increased the release of selected chemokines. Following carotid wire injury in athymic mice, injection of EPCs resulted not only in reduced neointima formation but also in altered cellular composition of the neointima with augmented accumulation of SMCs., Conclusion:  EPCs stimulate proliferation and migration of SMCs and increase their neointimal accumulation following vascular injury. Furthermore, EPCs context-dependently modify the SMC phenotype with protection from the transformative effect of cholesterol when a direct cell-cell contact is established., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Persistent inflammation following vascular injury drives neointimal hyperplasia (NIH). Specialized lipid mediators (SPM) mediate resolution which attenuates inflammation and downstream NIH. We investigated the effects of a synthetic analogue of resolvin D1 (RvD1) on vascular cells and in a model of rat carotid angioplasty., Methods: Human venous VSMC and endothelial cells (EC) were employed in migration, cell shape, toxicity, proliferation and p65 nuclear translocation assays. Murine RAW 264.7 cells were utilized to test the effect of pro-resolving compounds on phagocytic activity. A model of rat carotid angioplasty was used to evaluate the effects of 17R/S-benzo-RvD1 (benzo-RvD1) and 17R-RvD1 applied to the adventitia via 25% Pluronic gel. Immunostaining was utilized to examine Ki67 expression and leukocyte recruitment. Morphometric analysis was performed on arteries harvested 14 days after injury., Results: Exposure to benzo-RvD1 attenuated PDGF- stimulated VSMC migration across a range of concentrations (0.1-100 nM), similar to that observed with 17R-RvD1. Pre-treatment with either Benzo-RvD1 or 17R-RvD1 (10, 100nM) attenuated PDGF-BB-induced VSMC cytoskeletal changes to nearly baseline dimensions. Benzo-RvD1 demonstrated modest anti-proliferative activity on VSMC and EC at various concentrations, without significant cytotoxicity. Benzo-RvD1 (10nM) inhibited p65 nuclear translocation in cytokine-stimulated EC by 21% (p<0.05), similar to 17R-RvD1. Consistent with pro-resolving activities of other SPM, both 17R-RvD1 and benzo-RvD1 increased the phagocytic activity of RAW 264.7 cells against S. Aureus and Zymosan particles. There were no significant differences in Ki-67 or CD45 staining observed on day 3 after angioplasty. Periadventitial treatment with benzo-RvD1 reduced carotid neointimal area at 14 days compared to control (0.08 mm2 v. 0.18 mm2; p<0.05), with similar efficacy to 17R-RvD1., Conclusions: 17R/S-benzo-RvD1 and 17R-RvD1 exhibit similar pro-resolving and anti-migratory activity in cell-based assays, and both compounds attenuated NIH following acute arterial injury in rats. Further studies of the mechanisms of resolution following vascular injury, and the translational potential of SPM analogues, are indicated., Competing Interests: MSC - co-Founder of VasaRx and co-inventor of IP related to this work with the Regents of University of California and Brigham and Women’s Hospital. This does not alter our adherence to PLOS-One policies on sharing data and materials.

Aims: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance., Methods and Results: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvβ3 signalling abolished proliferation. However, VWF did not bind to αvβ3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvβ3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas., Conclusion: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Lower-extremity arterial disease is a major health problem with increasing prevalence, often leading to non-traumatic amputation, disability and mortality. The molecular mechanisms underpinning abnormal vascular wall remodeling are not fully understood. We hypothesized on the existence of a vascular tissue memory that may be transmitted through soluble signaling messengers, transferred from humans to healthy recipient animals, and consequently drive the recapitulation of arterial wall thickening and other vascular pathologies. We examined the effects of the intralesional infiltration for 6 days of arteriosclerotic popliteal artery-derived homogenates (100 µg of protein) into rats' full-thickness wounds granulation tissue. Animals infiltrated with normal saline solution or healthy brachial arterial tissue homogenate obtained from traumatic amputation served as controls. The significant thickening of arteriolar walls was the constant outcome in two independent experiments for animals receiving arteriosclerotic tissue homogenates. This material induced other vascular morphological changes including an endothelial cell phenotypic reprogramming that mirrored the donor's vascular histopathology. The immunohistochemical expression pattern of relevant vascular markers appeared to match between the human tissue and the corresponding recipient rats. These changes occurred within days of administration, and with no cross-species limitation. The identification of these "vascular disease drivers" may pave novel research avenues for atherosclerosis pathobiology.

Methylglyoxal (MGO) is an active metabolite of glucose and plays a prominent role in the pathogenesis of diabetic vascular complications, including endothelial cell apoptosis induced by oxidative stress. Metformin (MET), a widely prescribed antidiabetic agent, appears to reduce excessive reactive oxygen species (ROS) generation and limit cell apoptosis. However, the molecular mechanisms underlying this process are still not fully elucidated. We reported here that MET prevents MGO-induced apoptosis by suppressing oxidative stress in vitro and in vivo. Protein expression and protein phosphorylation were investigated using western blotting, ELISA, and immunohistochemical staining, respectively. Cell viability and apoptosis were assessed by the MTT assay, TUNEL staining, and Annexin V-FITC and propidium iodide double staining. ROS generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Our results revealed that MET prevented MGO-induced HUVEC apoptosis, inhibited apoptosis-associated biochemical changes such as loss of MMP, the elevation of the Bax/Bcl-2 ratio, and activation of cleaved caspase-3, and attenuated MGO-induced mitochondrial morphological alterations in a dose-dependent manner. MET pretreatment also significantly suppressed MGO-stimulated ROS production, increased signaling through the ROS-mediated PI3K/Akt and Nrf2/HO-1 pathways, and markedly elevated the levels of its downstream antioxidants. Finally, similar results were obtained in vivo, and we demonstrated that MET prevented MGO-induced oxidative damage, apoptosis, and inflammation. As expected, MET reversed MGO-induced downregulation of Nrf2 and p-Akt. In addition, a PI3K inhibitor (LY-294002) and a Nrf2 inhibitor (ML385) observably attenuated the protective effects of MET on MGO-induced apoptosis and ROS generation by inhibiting the Nrf2/HO-1 pathways, while a ROS scavenger (NAC) and a permeability transition pores inhibitor (CsA) completely reversed these effects. Collectively, these findings broaden our understanding of the mechanism by which MET regulates apoptosis induced by MGO under oxidative stress conditions, with important implications regarding the potential application of MET for the treatment of diabetic vascular complications., (© 2021. The Author(s).)

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis., (© 2021. The Author(s).)

Background: MiR-92a-3p and oxidative stress are associated with catheter-related thrombosis (CRT). As a kind of physical intervention, resistance exercise can effectively promote blood circulation. In this study, we investigated the roles of miR-92a-3p, oxidative stress and the P38 mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) pathway in CRT during resistance exercise., Methods: The rat CRT model was used for resistance exercise intervention. Moreover, pathological changes from the right jugular vein to the right auricle were observed under an electron microscope. In addition, reactive oxygen species (ROS) production, malondialdehyde (MDA) activity and heme oxygenase (HO-1) level in rat serum were detected via ELISA. The expression levels of miR-92A-3p and HO-1 in the vascular tissues of the rats were determined via real-time quantitative PCR. Additionally, the expression levels of HO-1, NF-κB P65, p38MAPK and IκBa in the venous tissues of the rats were analysed by Western blot analysis., Results: The pathological results showed that the thrombosis incidence rate in the CRT + RE group was lower than that in the CRT group. In the CRT group, the expression levels of ROS and MDA, which are markers related to oxidative stress in serum, significantly increased whilst the expression of HO-1 decreased. In the venous tissue, the expression of miR-92a-3p increased, the level of HO-1 decreased, the levels of p38MAPK and NF-κB p65 significantly increased but that of P-IκBa and IκBa significantly decreased. In the CRT + RE group, after administering the resistance exercise intervention, ROS production and MDA activity in serum significantly decreased, the expression level of HO-1 increased and the expression level of miR-92a-3p in the venous tissues significantly decreased and was negatively correlated with that of HO-1. The levels of p38MAPK and NF-κB p65 significantly decreased but that of P- IκBa and IκBa significantly increased., Conclusion: Resistance exercise intervention downregulated miR-92a-3p expression, repaired oxidative stress injury and prevented CRT formation., (© 2021. The Author(s).)

Background: Endothelial cell (EC) injury accelerates the progression of diabetic macrovascular complications. Hypoxia is an important cause of EC injury. Hypoxia-inducible factor-1 alpha (HIF-1α) is an important hypoxia regulatory protein. Our previous studies showed that high-glucose and hypoxic conditions could upregulate HIF-1α expression and enhance EC inflammatory injury, independently of the nuclear factor kappa-B (NF-κB) pathway. However, it is not clear whether HIF-1α plays a role in vascular disease through epigenetic-related mechanisms., Methods: We conducted gene expression analysis and molecular mechanistic studies in human umbilical vein endothelial cells (HUVECs) induced by hyperglycemia and hypoxia using RNA sequencing (RNA-seq) and small interfering HIF-1α (si-HIF-1α). We determined HIF-1α and Jumonji domain-containing protein 1 A (JMJD1A) expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, analyzed inflammatory protein secretion in the cell supernatant by enzymelinked immunosorbent assay (ELISA), and assessed protein interaction between HIF-1α and JMJD1A by chromatin immunoprecipitation (Ch-IP). We used the Cell Counting Kit8 (CCK-8) assay to analyze cell viability, and assessed oxidative stress indicators by using a detection kit and flow cytometry., Results: High glucose and hypoxia up-regulated HIF-1α expression, and down-regulated HIF-1α decreased the level of inflammation and oxidative stress in HUVECs. To determine the downstream pathways, we observed histone demethylases genes and related pathway by RNA-sEq. Among these, JMJD1A was the most upregulated gene in histone demethylases. Moreover, we observed that HIF-1α bound to the promoter of JMJD1A, and the ameliorative effects of si-HIF-1α on oxidative stress and inflammatory cytokines in high-glucose and hypoxia-induced HUVECs were reversed by JMJD1A overexpression. Furthermore, knockdown of JMJD1A decreased inflammatory and oxidative stress injury. To determine the JMJD1A-related factors, we conducted gene expression analysis on JMJD1A-knockdown HUVECs. We observed that downregulation of inflammation and the oxidative stress pathway were enriched and FOS and FOSB might be important protective transcription factors., Conclusions: These findings provide novel evidence that the HIF-1α/JMJD1A signaling pathway is involved in inflammation and oxidative stress in HUVECs induced by high glucose and hypoxia. Also, this pathway might act as a novel regulator of oxidative stress and inflammatory-related events in response to diabetic vascular injury and thus contribute to the pathological progression of diabetes and vascular disease., (© 2021. The Author(s).)

Objective: Arterial restenosis is the pathological narrowing of arteries after endovascular procedures, and it is an adverse event that causes patients to experience recurrent occlusive symptoms. Following angioplasty, vascular smooth muscle cells (SMCs) change their phenotype, migrate, and proliferate, resulting in neointima formation, a hallmark of arterial restenosis. SIKs (salt-inducible kinases) are a subfamily of the AMP-activated protein kinase family that play a critical role in metabolic diseases including hepatic lipogenesis and glucose metabolism. Their role in vascular pathological remodeling, however, has not been explored. In this study, we aimed to understand the role and regulation of SIK3 in vascular SMC migration, proliferation, and neointima formation., Approach and Results: We observed that SIK3 expression was low in contractile aortic SMCs but high in proliferating SMCs. It was also highly induced by growth medium in vitro and in neointimal lesions in vivo. Inactivation of SIKs significantly attenuated vascular SMC proliferation and up-regulated p21CIP1 and p27KIP1. SIK inhibition also suppressed SMC migration and modulated actin polymerization. Importantly, we found that inhibition of SIKs reduced neointima formation and vascular inflammation in a femoral artery wire injury model. In mechanistic studies, we demonstrated that inactivation of SIKs mainly suppressed SMC proliferation by down-regulating AKT (protein kinase B) and PKA (protein kinase A)-CREB (cAMP response element-binding protein) signaling. CRTC3 (CREB-regulated transcriptional coactivator 3) signaling likely contributed to SIK inactivation-mediated antiproliferative effects., Conclusions: These findings suggest that SIK3 may play a critical role in regulating SMC proliferation, migration, and arterial restenosis. This study provides insights into SIK inhibition as a potential therapeutic strategy for treating restenosis in patients with peripheral arterial disease.