Relationship between the extent of vascular injury and the evolution of surgically induced osteochondrosis lesions in a piglet model.

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.

Dynamic changes in proresolving lipid mediators and their receptors following acute vascular injury in male rats.

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.

Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

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.

Intervention of Asprosin Attenuates Oxidative Stress and Neointima Formation in Vascular Injury.

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.

Endothelial RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of ß-catenin and ILK signaling pathways after inflammatory vascular injury.

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 PaO2/FiO2 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 ß-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 ß-catenin and ILK signaling pathways after inflammatory vascular injury.

Cuproptosis and copper deficiency in ischemic vascular injury and repair.

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.

ZFP36L1 controls KLF16 mRNA stability in vascular smooth muscle cells during restenosis after vascular injury.

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-ß, 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.

Impact of Perivascular Adipose Tissue on Neointimal Formation Following Endovascular Placement.

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.

Potassium dehydroandrographolide succinate regulates the MyD88/CDH13 signaling pathway to enhance vascular injury-induced pathological vascular remodeling.

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.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia.

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.

Adventitial delivery of miR-145 to treat intimal hyperplasia post vascular injuries through injectable and in-situ self-assembling peptide hydrogels.

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.

Bone Morphogenetic Protein-4 Promotes Phenotypic Modulation via SMAD-4/MCT-4 Axis in Vascular Smooth Muscle Cells.

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.

A novel tiRNA-Glu-CTC induces nanoplastics accelerated vascular smooth muscle cell phenotypic switching and vascular injury through mitochondrial damage.

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.

CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

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.

miR-135a-5p overexpression in peripheral blood-derived exosomes mediates vascular injury in type 2 diabetes patients.

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.

The downstream network of STAT6 in promoting vascular smooth muscle cell phenotypic switch and neointimal formation.

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.

Role of Nuclear Receptor Subfamily 1 Group D Member 1 in the Proliferation, Migration of Vascular Smooth Muscle Cell, and Vascular Intimal Hyperplasia.

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.

Inflamed Adipose Tissue: Therapeutic Targets for Obesity-related Endothelial Injury.

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.

Why platelet mechanotransduction matters for hemostasis and thrombosis.

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.