Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries.

BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.

Transfer RNA-derived small RNA tRF-Glu-CTC attenuates neointimal formation via inhibition of fibromodulin.

Neointimal hyperplasia is a pathological vascular remodeling caused by abnormal proliferation and migration of subintimal vascular smooth muscle cells (VSMCs) following intimal injury. There is increasing evidence that tRNA-derived small RNA (tsRNA) plays an important role in vascular remodeling. The purpose of this study is to search for tsRNAs signature of neointima formation and to explore their potential functions. The balloon injury model of rat common carotid artery was replicated to induce intimal hyperplasia, and the differentially expressed tsRNAs (DE-tsRNAs) in arteries with intimal hyperplasia were screened by small RNA sequencing and tsRNA library. A total of 24 DE-tsRNAs were found in the vessels with intimal hyperplasia by small RNA sequencing. In vitro, tRF-Glu-CTC inhibited the expression of fibromodulin (FMOD) in VSMCs, which is a negative modulator of TGF-ß1 activity. tRF-Glu-CTC also increased VSMC proliferation and migration. In vivo experiments showed that inhibition of tRF-Glu-CTC expression after balloon injury of rat carotid artery can reduce the neointimal area. In conclusion, tRF-Glu-CTC expression is increased after vascular injury and inhibits FMOD expression in VSMCs, which influences neointima formation. On the other hand, reducing the expression of tRF-Glu-CTC after vascular injury may be a potential approach to prevent vascular stenosis.

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.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

PURPOSE: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. PROCEDURES: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. RESULTS: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. CONCLUSIONS: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

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.

Ginkgolide B Blocks Vascular Remodeling after Vascular Injury via Regulating Tgfß1/Smad Signaling Pathway.

Coronary artery disease (CAD) is the most prevalent cardiovascular disease worldwide, resulting in myocardial infarction (MI) and even sudden death. Following percutaneous coronary intervention (PCI), restenosis caused by vascular remodeling is always formed at the stent implantation site. Here, we show that Ginkgolide B (GB), a naturally occurring terpene lactone, effectively suppresses vascular remodeling and subsequent restenosis in wild-type mice following left carotid artery (LCA) injury. Additional experiments reveal that GB exerts a protective effect on vascular remodeling and further restenosis through modulation of the Tgfß1/Smad signaling pathway in vivo and in human vascular smooth muscle cells (HVSMAs) but not in human umbilical vein endothelial cells (HUVECs) in vitro. Moreover, the beneficial effect of GB is abolished after incubated with pirfenidone (PFD, a drug for idiopathic pulmonary fibrosis, IPF), which can inhibit Tgfß1. In Tgfß1-/- mice, treatment with pirfenidone capsules and Yinxingneizhi Zhusheye (including Ginkgolide B) fails to improve vascular remodeling and restenosis. In conclusion, our data identify that GB could be a potential novel therapeutic agent to block vessel injury-associated vascular remodeling and further restenosis and show significant repression of Tgfß1/Smad signaling pathway.

Severe arterial injury heals with a complex clonal structure involving a large fraction of surviving smooth muscle cells.

BACKGROUND AND AIMS: Smooth muscle cell (SMC) lineage cells in atherosclerosis and flow cessation-induced neointima are oligoclonal, being recruited from a tiny fraction of medial SMCs that modulate and proliferate. The present study aimed to investigate the clonal structure of SMC lineage cells healing more severe arterial injury. METHODS: Arterial injury (wire, stretch, and partial ligation) was inflicted on the right carotid artery in mice with homozygous, SMC-restricted, stochastically recombining reporter transgenes that produced mosaic expression of 10 distinguishable fluorescent phenotypes for clonal tracking. Healed arteries and contra-lateral controls were analyzed after 3 weeks. Additional analysis of cell death and proliferation after injury was performed in wildtype mice. RESULTS: The total number of SMC lineage cells in healed arteries was comparable to normal arteries but comprised significantly fewer fluorescent phenotypes. The population had a complex, intermixed, clonal structure. By statistical analysis of expected versus observed fractions of fluorescent phenotypes and visual inspection of coherent groups of same-colored cells, we concluded that >98% of SMC lineage cells in healed arteries belonged to a detectable clone, indicating that nearly all surviving SMCs after severe injury at some point undergo proliferation. This was consistent with serial observations in the first week after injury, which showed severe loss of medial cells followed by widespread proliferation. CONCLUSIONS: After severe arterial injury, many surviving SMCs proliferate to repair the media and form a neointima. This indicates that the fraction of medial SMCs that are mobilized to repair arteries increases with the level of injury.

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.

Tartary buckwheat protein-derived peptide AFYRW alleviates H2O2-induced vascular injury via the PI3K/AKT/NF-κB pathway.

Tartary buckwheat protein-derived peptide (Ala-Phe-Tyr-Arg-Trp, AFYRW) is a natural active peptide that hampers the atherosclerosis process, but the underlying role of AFYRW in angiogenesis remains unknown. Here, we present a system-based study to evaluate the effects of AFYRW on H2O2-induced vascular injury in human umbilical vein endothelial cells (HUVECs). HUVECs were co-incubated with H2O2 for 2 h in the vascular injury model, and AFYRW was added 24 h in advance to investigate the protective mechanism of vascular injury. We identified that AFYRW inhibits oxidative stress, cell migration, cell invasion, and angiogenesis in H2O2-treated HUVECs. In addition, we found H2O2-induced upregulation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), phosphorylation of nuclear factor-κB (NF-κB) p65 and nuclear translocation of NF-κB decreased by AFYRW. Taken together, AFYRW attenuated H2O2-induced vascular injury through the PI3K/AKT/NF-κB pathway. Thereby, AFYRW may serve as a therapeutic option for vascular injuries.

Extracellular Matrix Remodeling in Vascular Disease: Defining Its Regulators and Pathological Influence.

Because of structural and cellular differences (ie, degrees of matrix abundance and cross-linking, mural cell density, and adventitia), large and medium-sized vessels, in comparison to capillaries, react in a unique manner to stimuli that induce vascular disease. A stereotypical vascular injury response is ECM (extracellular matrix) remodeling that occurs particularly in larger vessels in response to injurious stimuli, such as elevated angiotensin II, hyperlipidemia, hyperglycemia, genetic deficiencies, inflammatory cell infiltration, or exposure to proinflammatory mediators. Even with substantial and prolonged vascular damage, large- and medium-sized arteries, persist, but become modified by (1) changes in vascular wall cellularity; (2) modifications in the differentiation status of endothelial cells, vascular smooth muscle cells, or adventitial stem cells (each can become activated); (3) infiltration of the vascular wall by various leukocyte types; (4) increased exposure to critical growth factors and proinflammatory mediators; and (5) marked changes in the vascular ECM, that remodels from a homeostatic, prodifferentiation ECM environment to matrices that instead promote tissue reparative responses. This latter ECM presents previously hidden matricryptic sites that bind integrins to signal vascular cells and infiltrating leukocytes (in coordination with other mediators) to proliferate, invade, secrete ECM-degrading proteinases, and deposit injury-induced matrices (predisposing to vessel wall fibrosis). In contrast, in response to similar stimuli, capillaries can undergo regression responses (rarefaction). In summary, we have described the molecular events controlling ECM remodeling in major vascular diseases as well as the differential responses of arteries versus capillaries to key mediators inducing vascular injury.

LINC00346 regulates NLRP1-mediated pyroptosis and autophagy via binding to microRNA-637 in vascular endothelium injury.

Endothelial injury and dysfunction contributes to atherosclerosis. LINC00346 plays a key role in vascular endothelial cell injury, however, the specific mechanism remains unclear. This study intends to further explore the relationship between LINC00346 and vascular endothelial injury. Circulating LINC00346 was significantly elevated in patients with coronary artery disease and had high diagnostic value for coronary artery disease. In cell experiments, we found that LINC00346 expression was significantly increased in the oxidized low-density lipoprotein (ox-LDL) intervention group, and LINC00346 knockdown delayed ox-LDL induced human umbilical vein endothelial cell (HUVEC) endothelial-to-mesenchymal transition. In addition, knockdown of LINC00346 mitigated ox-LDL-induced NOD-like receptor protein 1 (NLRP1)-mediated inflammasome formation and pyroptosis, but had no significant effect on NLRP3. By observing the number of autophagosome and detecting intracellular autophagic flux, we found that LINC00346 knockdown inhibited the ox-LDL-induced increase in intracellular autophagy level. Dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA-pull down assay were performed to confirm the inter-molecular interaction. LINC00346 acted as microRNA-637 sponge to up-regulate the expression of NLRP1. Up-regulation of microRNA-637 alleviated NLRP1-mediated pyroptosis in HUVEC and reduced intracellular autophagosome and autolysosome formation. Finally, we explored whether pyropotosis and autophagy interact with each other. We found that inhibition of intracellular autophagy could alleviate NLRP1-mediated pyroptosis. In conclusion, LINC00346 inhibited the activation of NLRP1-mediated pyroptosis and autophagy via binding to microRNA-637, therefore mitigating vascular endothelial injury.

CD1d-Dependent Natural Killer T Cells Mediate Hypertension and Vascular Injury Through Interleukin-17A.

Background Different T-lymphocyte subsets, including CD1d-dependent natural killer T (NKT) cells, play distinct roles in hypertension, highlighting the importance of identifying key immune cells for its treatment. This study aimed to determine the unknown effects of CD1d-dependent NKT cells on hypertension and vascular injury. Methods and Results Hypertension models were induced in male CD1d knockout (CD1dko), wild-type, and adoptive bone marrow transfer mice by angiotensin II (Ang II) or deoxycorticosterone acetate salt. Blood pressure was measured by the tail-cuff system and radiotelemetry. Vascular injury was assessed by histologic studies or aortic ring assay. Inflammation was detected by flow cytometry, quantitative real-time polymerase chain reaction, or ELISA. Results showed that Ang II infusion significantly reduced CD1d expression and NKT cell numbers in the aorta of mice. CD1dko mice exhibited worsened blood pressure elevation, vascular injury, and inflammatory response induced by Ang II or deoxycorticosterone acetate salt. However, these effects were markedly reversed in wild-type mice treated with NKT cell-specific activator. Adoptive transfer of CD1dko bone marrow cells to wild-type mice also significantly worsened Ang II-induced responses. Mechanistically, CD1dko increased Ang II-induced interleukin-6 production and activated signal transducer and activator of transcription 3 and orphan nuclear receptor γ, subsequently inducing interleukin-17A production. Neutralizing interleukin-17A partially reversed Ang II-induced hypertension and vascular injury in CD1dko mice. In addition, levels of NKT cells were lower in the blood of patients with hypertension (n=57) compared with normotensive individuals (n=87). Conclusions These findings reveal a previously unknown role for CD1d-dependent NKT cells in hypertension and vascular injury, indicating that NKT cell activation could be a promising therapeutic target for hypertension.

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.

Cathepsin S inhibition in dendritic cells prevents Th17 cell differentiation in perivascular adipose tissues following vascular injury in diabetic rats.

In the context of diabetes mellitus (DM), the circulating cathepsin S (CTSS) level is significantly higher in the cardiovascular disease group. Therefore, this study was designed to investigate the role of CTSS in restenosis following carotid injury in diabetic rats. To induce DM, 60 mg/kg of streptozotocin (STZ) in citrate buffer was injected intraperitoneally into Sprague-Dawley rats. After successful modeling of DM, wire injury of the rat carotid artery was performed, followed by adenovirus transduction. Levels of blood glucose and Th17 cell surface antigens including ROR-γt, IL-17A, IL-17F, IL-22, and IL-23 in perivascular adipose tissues (PVAT) were evaluated. For in vitro analysis, human dendritic cells (DCs) were treated with 5.6-25 mM glucose for 24 h. The morphology of DCs was observed using an optical microscope. CD4+ T cells derived from human peripheral blood mononuclear cells were cocultured with DCs for 5 days. Levels of IL-6, CTSS, ROR-γt, IL-17A, IL-17F, IL-22 and IL-23 were measured. Flow cytometry was conducted to detect DC surface biomarkers (CD1a, CD83, and CD86) and Th17 cell differentiation. The collected DCs presented a treelike shape and were positive for CD1a, CD83, and CD86. Glucose impaired DC viability at the dose of 35 mM. Glucose treatment led to an increase in CTSS and IL-6 expression in DCs. Glucose-treated DCs promoted the differentiation of Th17 cells. CTSS depletion downregulated IL-6 expression and inhibited Th17 cell differentiation in vitro and in vivo. CTSS inhibition in DCs inhibits Th17 cell differentiation in PVAT tissues from diabetic rats following vascular injury.

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.

Paeonol Promotes Reendothelialization After Vascular Injury Through Activation of c-Myc/VEGFR2 Signaling Pathway.

Purpose: Dysfunction of endothelium is associated with multiple pathological vascular diseases. However, how to regulate reendothelialization after vascular injury is not well defined. This study aims to determine whether and how Paeonol controls reendothelialization following artery injury. Methods: The endothelium of murine carotid artery was denuded by catheter guide wires injury. H&E staining and IF staining were performed to determine whether Paeonol is critical for reendothelialization. BRDU Incorporation Assay, Boyden Chamber Migration Assay, Tube Formation Assay, and Spheroid Sprouting Assay were used to investigate whether Paeonol is involved in regulating proliferation and migration of endothelial cells. The underlying mechanism of how Paeonol regulates reendothelialization was determined by Molecular docking simulation and CO-IP Assay. Results: Paeonol treatment significantly inhibits neointima formation in carotid artery ligation model by promoting proliferation and migration of endothelial cells. Mechanistically, Paeonol enhances c-Myc expression, consequently interacts with VEGFR2 results in activating VEGF signaling pathway, and eventually promotes reendothelialization after vascular injury. Conclusion: Our data demonstrated that Paeonol plays a critical role in regulating vascular reendothelialization, which may be therapeutically used for treatment of pathological vascular diseases.

Tubulovascular protection from protease-activated receptor-1 depletion during AKI-to-CKD transition.

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-ß/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.

Redistribution of the chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and vascular fibrosis.

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-ß1 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-ß1-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-ß1 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.