Loss of Smooth Muscle Tenascin-X Inhibits Vascular Remodeling Through Increased TGF-ß Signaling.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.

Blockade of CD47 function attenuates restenosis by promoting smooth muscle cell efferocytosis and inhibiting their migration and proliferation.

Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cß3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin ß3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia.

Mast cell activation and degranulation in acute artery injury: A target for post-operative therapy.

The increasing incidence of cardiovascular disease (CVD) has led to a significant ongoing need to address this surgically through coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI). From this, there continues to be a substantial burden of mortality and morbidity due to complications arising from endothelial damage, resulting in restenosis. Whilst mast cells (MC) have been shown to have a causative role in atherosclerosis and other vascular diseases, including restenosis due to vein engraftment; here, we demonstrate their rapid response to arterial wire injury, recapitulating the endothelial damage seen in PCI procedures. Using wild-type mice, we demonstrate accumulation of MC in the femoral artery post-acute wire injury, with rapid activation and degranulation, resulting in neointimal hyperplasia, which was not observed in MC-deficient KitW-sh/W-sh mice. Furthermore, neutrophils, macrophages, and T cells were abundant in the wild-type mice area of injury but reduced in the KitW-sh/W-sh mice. Following bone-marrow-derived MC (BMMC) transplantation into KitW-sh/W-sh mice, not only was the neointimal hyperplasia induced, but the neutrophil, macrophage, and T-cell populations were also present in these transplanted mice. To demonstrate the utility of MC as a target for therapy, we administered the MC stabilizing drug, disodium cromoglycate (DSCG) immediately following arterial injury and were able to show a reduction in neointimal hyperplasia in wild-type mice. These studies suggest a critical role for MC in inducing the conditions and coordinating the detrimental inflammatory response seen post-endothelial injury in arteries undergoing revascularization procedures, and by targeting the rapid MC degranulation immediately post-surgery with DSCG, this restenosis may become a preventable clinical complication.

PHB2 Maintains the Contractile Phenotype of VSMCs by Counteracting PKM2 Splicing.

BACKGROUND: Phenotypic transition of vascular smooth muscle cells (VSMCs) accounts for the pathogenesis of a variety of vascular diseases during the early stage. Recent studies indicate the metabolic reprogramming may be involved in VSMC phenotypic transition. However, the definite molecules that link energy metabolism to distinct VSMC phenotype remain elusive. METHODS: A carotid artery injury model was used to study postinjury neointima formation as well as VSMC phenotypic transition in vivo. RNA-seq analysis, cell migration assay, collagen gel contraction assay, wire myography assay, immunoblotting, protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: We collected cell energy-regulating genes by using Gene Ontology annotation and applied RNA-Seq analysis of transforming growth factor-ß or platelet-derived growth factor BB stimulated VSMCs. Six candidate genes were overlapped from energy metabolism-related genes and genes reciprocally upregulated by transforming growth factor-ß and downregulated by platelet-derived growth factor BB. Among them, prohibitin 2 has been reported to regulate mitochondrial oxidative phosphorylation. Indeed, prohibitin 2-deficient VSMCs lost the contractile phenotype as evidenced by reduced contractile proteins. Consistently, Phb2SMCKO mice were more susceptible to postinjury VSMC proliferation and neointima formation compared with Phb2flox/flox mice. Further protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay revealed that prohibitin 2, through its C-terminus, directly interacts with hnRNPA1, a key modulator of pyruvate kinase M1/2 (PKM) mRNA splicing that promotes PKM2 expression and glycolysis. Prohibitin 2 deficiency facilitated PKM1/2 mRNA splicing and reversion from PKM1 to PKM2, and enhanced glycolysis in VSMCs. Blocking prohibitin 2-hnRNPA1 interaction resulted in increased PKM2 expression, enhanced glycolysis, repressed contractile marker genes expression in VSMCs, as well as aggravated postinjury neointima formation in vivo. CONCLUSIONS: Prohibitin 2 maintains VSMC contractile phenotype by interacting with hnRNPA1 to counteract hnRNPA1-mediated PKM alternative splicing and glucose metabolic reprogramming.

Matrix-Binding, N-Cadherin-Targeting Chimeric Peptide Inhibits Intimal Thickening but Not Endothelial Repair in Balloon-Injured Carotid Arteries.

BACKGROUND: Treatment of occluded vessels can involve angioplasty, stenting, and bypass grafting, which can be limited by restenosis and thrombosis. Drug-eluting stents attenuate restenosis, but the current drugs used are cytotoxic, causing smooth muscle cell (SMC) and endothelial cell (EC) death that may lead to late thrombosis. N-cadherin is a junctional protein expressed by SMCs, which promotes directional SMC migration contributing to restenosis. We propose that engaging N-cadherin with mimetic peptides can act as a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs. METHODS: We designed a novel N-cadherin-targeting chimeric peptide with a histidine-alanine-valine cadherin-binding motif, combined with a fibronectin-binding motif from Staphylococcus aureus. This peptide was tested in SMC and EC culture assays of migration, viability, and apoptosis. Rat carotid arteries were balloon injured and treated with the N-cadherin peptide. RESULTS: Treating scratch-wounded SMCs with the N-cadherin-targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide colocalized with fibronectin. Importantly, EC junction, permeability, or migration was not impacted by peptide treatment in vitro. We also demonstrated that the chimeric peptide persisted for 24 hours after transient delivery in the balloon-injured rat carotid artery. Treatment with the N-cadherin-targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury. Reendothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment. CONCLUSIONS: These studies show that an N-cadherin-binding and fibronectin-binding chimeric peptide is effective in inhibiting SMC migration in vitro and in vivo and limiting neointimal hyperplasia after balloon angioplasty without affecting EC repair. These results establish the potential of an advantageous SMC-selective strategy for antirestenosis therapy.

Glycolysis and de novo fatty acid synthesis cooperatively regulate pathological vascular smooth muscle cell phenotypic switching and neointimal hyperplasia.

Switching of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a dedifferentiated (proliferative) phenotype contributes to neointima formation, which has been demonstrated to possess a tumor-like nature. Dysregulated glucose and lipid metabolism is recognized as a hallmark of tumors but has not thoroughly been elucidated in neointima formation. Here, we investigated the cooperative role of glycolysis and fatty acid synthesis in vascular injury-induced VSMC dedifferentiation and neointima formation. We found that the expression of hypoxia-inducible factor-1α (HIF-1α) and its target 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a critical glycolytic enzyme, were induced in the neointimal VSMCs of human stenotic carotid arteries and wire-injured mouse carotid arteries. HIF-1α overexpression led to elevated glycolysis and resulted in a decreased contractile phenotype while promoting VSMC proliferation and activation of the mechanistic target of rapamycin complex 1 (mTORC1). Conversely, silencing Pfkfb3 had the opposite effects. Mechanistic studies demonstrated that glycolysis generates acetyl coenzyme A to fuel de novo fatty acid synthesis and mTORC1 activation. Whole-transcriptome sequencing analysis confirmed the increased expression of PFKFB3 and fatty acid synthetase (FASN) in dedifferentiated VSMCs. More importantly, FASN upregulation was observed in neointimal VSMCs of human stenotic carotid arteries. Finally, interfering with PFKFB3 or FASN suppressed vascular injury-induced mTORC1 activation, VSMC dedifferentiation, and neointima formation. Together, this study demonstrated that PFKFB3-mediated glycolytic reprogramming and FASN-mediated lipid metabolic reprogramming are distinctive features of VSMC phenotypic switching and could be potential therapeutic targets for treating vascular diseases with neointima formation. © 2023 The Pathological Society of Great Britain and Ireland.

Transcription Factor 21: A Transcription Factor That Plays an Important Role in Cardiovascular Disease.

BACKGROUND: According to the World Health Organisation's Health Report 2019, approximately 17.18 million people die from cardiovascular disease each year, accounting for more than 30% of all global deaths. Therefore, the occurrence of cardiovascular disease is still a global concern. The transcription factor 21 (TCF21) plays an important role in cardiovascular diseases. This article reviews the regulation mechanism of TCF21 expression and activity and focuses on its important role in atherosclerosis in order to contribute to the development of diagnosis and treatment of cardiovascular diseases. SUMMARY: TCF21 is involved in the phenotypic regulation of vascular smooth muscle cells (VSMCs), promotes the proliferation and migration of VSMCs, and participates in the activation of inflammatory sequences. Increased proliferation and migration of VSMCs can lead to neointimal hyperplasia after vascular injury. Abnormal hyperplasia of neointima and inflammation are one of the main features of atherosclerosis. Therefore, targeting TCF21 may become a potential treatment for relieving atherosclerosis. KEY MESSAGES: TCF21 as a member of basic helix-loop-helix transcription factors regulates cell growth and differentiation by modulating gene expression during the development of different organs and plays an important role in cardiovascular development and disease. VSMCs and cells derived from VSMCs constitute the majority of plaques in atherosclerosis. TCF21 plays a key role in regulation of VSMCs' phenotype, thus accelerating atherogenesis in the early stage. However, TCF21 enhances plaque stability in late-stage atherosclerosis. The dual role of TCF21 should be considered in the translational medicine.

Preliminary studies on intimal injury related to stent retrieval in a canine model.

OBJECTIVE: This pilot study aimed to observe intimal injuries related to stent retrieval in the iliac artery of a canine. BACKGROUND: In-stent restenosis remains challenging owing to permanent stent implantation. A retrievable stent may be alternative for intervention without permanent residue. METHODS: Five retrievable stents with point-to-point overlapped double-layer scaffolds were deployed into the iliac arteries and retrieved on days 14, 21, 28, 35, and 42 from five canines. RESULTS: Arterial diameter decreased by 9-10% before retrieval and 15% on day 14 after retrieval. In the 14-day-stent, the stent surface was clean without visible fibrin. In the 28-day-stent, the overlay was mainly composed of fibrin and fibroblasts. The proliferation of smooth muscle cells has not yet been observed with α-smooth muscle actin staining. In the 42-day-stent, endothelial and smooth muscle cells decreased under the struts, and the internal elastic lamina was interrupted segmentally. Neointima formation involves fibroblasts and smooth muscle cells. Neointimal thickness was negatively correlated with strut space. Stent traces on the artery wall tended to be flat at a follow-up14 days after retrieval. The primary intima was completely covered by neointima. Two stents could not be retrieved because of in-stent thrombosis or capture loss. CONCLUSIONS: The stent was covered mainly by depositional fibrin after 28 days and by typical neointima after 42 days. The stent retrieval procedure did not induce injury to vascular smooth muscle, and the intima repair was performed 14 days after stent retrieval.

Topographic distribution of inflammation factors in a healing aneurysm.

BACKGROUND: Healing of intracranial aneurysms following endovascular treatment relies on the organization of early thrombus into mature scar tissue and neointima formation. Activation and deactivation of the inflammation cascade plays an important role in this process. In addition to timely evolution, its topographic distribution is hypothesized to be crucial for successful aneurysm healing. METHODS: Decellularized saccular sidewall aneurysms were created in Lewis rats and coiled. At follow-up (after 3 days (n = 16); 7 days (n = 19); 21 days (n = 8)), aneurysms were harvested and assessed for healing status. In situ hybridization was performed for soluble inflammatory markers (IL6, MMP2, MMP9, TNF-α, FGF23, VEGF), and immunohistochemical analysis to visualize inflammatory cells (CD45, CD3, CD20, CD31, CD163, HLA-DR). These markers were specifically documented for five regions of interest: aneurysm neck, dome, neointima, thrombus, and adjacent vessel wall. RESULTS: Coiled aneurysms showed enhanced patterns of thrombus organization and neointima formation, whereas those without treatment demonstrated heterogeneous patterns of thrombosis, thrombus recanalization, and aneurysm growth (p = 0.02). In coiled aneurysms, inflammation markers tended to accumulate inside the thrombus and in the neointima (p < 0.001). Endothelial cells accumulated directly in the neointima (p < 0.0001), and their presence was associated with complete aneurysm healing. CONCLUSION: The presence of proinflammatory cells plays a crucial role in aneurysm remodeling after coiling. Whereas thrombus organization is hallmarked by a pronounced intra-thrombotic inflammatory reaction, neointima maturation is characterized by direct invasion of endothelial cells. Knowledge concerning topographic distribution of regenerative inflammatory processes may pave the way for future treatment modalities which enhance aneurysm healing after endovascular therapy.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

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.

YAP and TAZ in Vascular Smooth Muscle Confer Protection Against Hypertensive Vasculopathy.

BACKGROUND: Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. We hypothesize that appropriate mechanotransduction and contractile function in vascular smooth muscle cells are crucial to maintain vascular wall integrity. The Hippo pathway effectors YAP (yes-associated protein 1) and TAZ (WW domain containing transcription regulator 1) have been identified as mechanosensitive transcriptional coactivators. However, their role in vascular smooth muscle cell mechanotransduction has not been investigated in vivo. METHODS: We performed physiological and molecular analyses utilizing an inducible smooth muscle-specific YAP/TAZ knockout mouse model. RESULTS: Arteries lacking YAP/TAZ have reduced agonist-mediated contraction, decreased myogenic response, and attenuated stretch-induced transcriptional regulation of smooth muscle markers. Moreover, in established hypertension, YAP/TAZ knockout results in severe vascular lesions in small mesenteric arteries characterized by neointimal hyperplasia, elastin degradation, and adventitial thickening. CONCLUSIONS: This study demonstrates a protective role of YAP/TAZ against hypertensive vasculopathy.

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

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.

Sulfamethizole attenuates poloxamer 407-induced atherosclerotic neointima formation via inhibition of mTOR in C57BL/6 mice.

Mammalian target of Rapamycin C1 (mTORC1) inhibition limits plaque progression in atherosclerosis. The present study evaluated the protective effect of sulfamethizole on poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition. Poloxamer 407 (P-407) (0.5 g/kg body weight) was administered intraperitoneally to male C57BL/6 mice every third day for 148 days to induce chronic hyperlipidemia. From Day 121 to 148, animals were additionally administered Sulfamethizole (5, 10, and 50 mg/kg, p.o.), Rapamycin (0.5 mg/kg, positive control), or vehicle (1 ml/kg). Plasma lipid levels were measured on Days 120 and 148. Upon sacrifice, histological studies were performed, and aortic tissue interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and mTOR levels were evaluated. A molecular docking study was carried out to mimic the interaction of sulfamethizole with mTOR protein. Chronic P-407 administration significantly (p < 0.001) elevated plasma lipid levels, compared with those of the normal control group. Chronic hyperlipidemia resulted in increased tunica intima thickness, collagen deposition, and IL-6, TNF-α, and mTOR levels. Treatment with Sulfamethizole attenuated these parameters significantly in a dose-dependent manner. Molecular docking studies showed a significant interaction of Sulfamethizole with mTOR. In conclusion, this study suggests that sulfamethizole significantly limits poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition.

Sodium tanshinone IIA sulfonate ameliorates neointima by protecting endothelial progenitor cells in diabetic mice.

BACKGROUND: Endothelial progenitor cells (EPCs) transplantation is one of the effective therapies for neointima associated with endothelial injury. Diabetes impairs the function of EPCs and cumbers neointima prevention of EPC transplantation with an ambiguous mechanism. Sodium Tanshinone IIA Sulfonate (STS) is an endothelium-protective drug but whether STS protects EPCs in diabetes is still unknown. METHODS: EPCs were treated with High Glucose (HG), STS, and Nucleotide-binding Domain-(NOD) like Receptor 3 (NLRP3), caspase-1, the Receptor of Advanced Glycation End products (AGEs) (RAGE) inhibitors, Thioredoxin-Interacting Protein (TXNIP) siRNA, and EPC proliferation, differentiation functions, and senescence were detected. The treated EPCs were transplanted into db/db mice with the wire-injured Common Carotid Artery (CCA), and the CD31 expression and neointima were detected in the CCA inner wall. RESULTS: We found that STS inhibited HG-induced expression of NLRP3, the production of active caspase-1 (p20) and mature IL-1ß, the expression of catalase (CAT) cleavage, γ-H2AX, and p21 in EPCs. STS restored the expression of Ki67, CD31 and von Willebrand Factor (vWF) in EPCs; AGEs were found in the HG-treated EPCs supernatant, and RAGE blocking inhibited the expression of TXNIP and the production of p20, which was mimicked by STS. STS recovered the expression of CD31 in the wire-injured CCA inner wall and the prevention of neointima in diabetic mice with EPCs transplantation. CONCLUSION: STS inhibits the aggravated neointima hyperplasia by protecting the proliferation and differentiation functions of EPC and inhibiting EPC senescence in diabetic mice. The mechanism is related to the preservation of CAT activity by inhibiting the RAGE-TXNIP-NLRP3 inflammasome pathway.

Intimal growth on the luminal surface of arteriovenous grafts in rats.

BACKGROUND: Endothelial cells are known to grow on the luminal surface of arteriovenous grafts (AVGs) used in hemodialysis. Although endothelial cells are important for preventing infection, a detailed growth of endothelial cells in AVGs is unknown. This study sought to create a simpler animal model of AVGs and to investigate how endothelial cells form on the luminal surface. METHODS: Polyethylene grafts were placed between the cervical artery and vein of Wistar rats. The grafts were removed at 6 h, 24 h, 3 days, or 7 days after placement. The luminal surface was observed under optical and polarizing microscopy and stained with endothelial cell markers (LEL, CD31), the progenitor cell marker CD34, and the macrophage marker ED-1. RESULTS: Microscopy demonstrated many diffuse vascular endothelial cells on the luminal surface of AVGs after placement. While there was no difference in the number of LEL-positive cells between the arterial side (AS) and venous side (VS) at 6 h or 7 days, there were significantly more of these cells on the VS at both 24 h and 3 days (p < 0.05). Analysis at 24 h showed some CD31-positive cells and few CD34-positive cells. CONCLUSIONS: This was the first study to use a simple rat model of AVG placement. Endothelial cell formation was initially more active on the VS than on the AS, but these cells subsequently increased in number across the luminal surface. Future clinical studies might contribute clinically by confirming whether AS versus VS puncture results in different infection rates.

Increased expression of the P2Y12 receptor is involved in the failure of autogenous arteriovenous fistula caused by stenosis.

OBJECTIVE: This study investigated the role of the P2Y12 receptor in autogenous arteriovenous fistula (AVF) failure resulting from stenosis. METHODS: Stenotic venous tissues and blood samples were obtained from patients with end-stage renal disease (ESRD) together with AVF stenosis, while venous tissues and blood samples were collected from patients with ESRD undergoing initial AVF surgery as controls. Immunohistochemistry and/or immunofluorescence techniques were utilized to assess the expression of P2Y12, transforming growth factor-ß1 (TGF-ß1), monocyte chemotactic protein 1 (MCP-1), and CD68 in the venous tissues. The expression levels of P2Y12, TGFß1, and MCP-1 were quantified using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Double and triple immunofluorescence staining was performed to precisely localize the cellular localization of P2Y12 expression. RESULTS: Expression levels of P2Y12, TGFß1, MCP-1, and CD68 were significantly higher in stenotic AVF venous tissues than in the control group tissues. Double and triple immunofluorescence staining of stenotic AVF venous tissues indicated that P2Y12 was predominantly expressed in α-SMA-positive vascular smooth muscle cells (VSMCs) and, to a lesser extent, in CD68-positive macrophages, with limited expression in CD31-positive endothelial cells. Moreover, a subset of macrophage-like VSMCs expressing P2Y12 were observed in both stenotic AVF venous tissues and control venous tissues. Additionally, a higher number of P2Y12+/TGF-ß1+ double-positive cells were identified in stenotic AVF venous tissues than in the control group tissues. CONCLUSION: Increased expression of P2Y12 in stenotic AVF venous tissues of patients with ESRD suggests its potential involvement in the pathogenesis of venous stenosis within AVFs.

BMPER Improves Vascular Remodeling and the Contractile Vascular SMC Phenotype.

Dedifferentiated vascular smooth muscle cells (vSMCs) play an essential role in neointima formation, and we now aim to investigate the role of the bone morphogenetic protein (BMP) modulator BMPER (BMP endothelial cell precursor-derived regulator) in neointima formation. To assess BMPER expression in arterial restenosis, we used a mouse carotid ligation model with perivascular cuff placement. Overall BMPER expression after vessel injury was increased; however, expression in the tunica media was decreased compared to untreated control. Consistently, BMPER expression was decreased in proliferative, dedifferentiated vSMC in vitro. C57BL/6_Bmper+/- mice displayed increased neointima formation 21 days after carotid ligation and enhanced expression of Col3A1, MMP2, and MMP9. Silencing of BMPER increased the proliferation and migration capacity of primary vSMCs, as well as reduced contractibility and expression of contractile markers, whereas stimulation with recombinant BMPER protein had the opposite effect. Mechanistically, we showed that BMPER binds insulin-like growth factor-binding protein 4 (IGFBP4), resulting in the modulation of IGF signaling. Furthermore, perivascular application of recombinant BMPER protein prevented neointima formation and ECM deposition in C57BL/6N mice after carotid ligation. Our data demonstrate that BMPER stimulation causes a contractile vSMC phenotype and suggest that BMPER has the potential for a future therapeutic agent in occlusive cardiovascular diseases.

Inhibition of p90RSK Ameliorates PDGF-BB-Mediated Phenotypic Change of Vascular Smooth Muscle Cell and Subsequent Hyperplasia of Neointima.

Platelet-derived growth factor type BB (PDGF-BB) regulates vascular smooth muscle cell (VSMC) migration and proliferation, which play critical roles in the development of vascular conditions. p90 ribosomal S6 kinase (p90RSK) can regulate various cellular processes through many different target substrates in several cell types, but the regulatory function of p90RSK on PDGF-BB-mediated cell migration and proliferation and subsequent vascular neointima formation has not yet been extensively examined. In this study, we investigated whether p90RSK inhibition protects VSMCs against PDGF-BB-induced cellular phenotypic changes and the molecular mechanisms underlying the effect of p90RSK inhibition on neointimal hyperplasia in vivo. Pretreatment of cultured primary rat VSMCs with FMK or BI-D1870, which are specific inhibitors of p90RSK, suppressed PDGF-BB-induced phenotypic changes, including migration, proliferation, and extracellular matrix accumulation, in VSMCs. Additionally, FMK and BI-D1870 repressed the PDGF-BB-induced upregulation of cyclin D1 and cyclin-dependent kinase-4 expression. Furthermore, p90RSK inhibition hindered the inhibitory effect of PDGF-BB on Cdk inhibitor p27 expression, indicating that p90RSK may induce VSMC proliferation by regulating the G0/G1 phase. Notably, treatment with FMK resulted in attenuation of neointima development in ligated carotid arteries in mice. The findings imply that p90RSK inhibition mitigates the phenotypic switch and neointimal hyperplasia induced by PDGF-BB.

Elastin, arterial mechanics, and stenosis.

Elastin is a long-lived extracellular matrix protein that is organized into elastic fibers that provide elasticity to the arterial wall, allowing stretch and recoil with each cardiac cycle. By forming lamellar units with smooth muscle cells, elastic fibers transduce tissue-level mechanics to cell-level changes through mechanobiological signaling. Altered amounts or assembly of elastic fibers leads to changes in arterial structure and mechanical behavior that compromise cardiovascular function. In particular, genetic mutations in the elastin gene (ELN) that reduce elastin protein levels are associated with focal arterial stenosis, or narrowing of the arterial lumen, such as that seen in supravalvular aortic stenosis and Williams-Beuren syndrome. Global reduction of Eln levels in mice allows investigation of the tissue- and cell-level arterial mechanical changes and associated alterations in smooth muscle cell phenotype that may contribute to stenosis formation. A loxP-floxed Eln allele in mice highlights cell type- and developmental origin-specific mechanobiological effects of reduced elastin amounts. Eln production is required in distinct cell types for elastic layer formation in different parts of the mouse vasculature. Eln deletion in smooth muscle cells from different developmental origins in the ascending aorta leads to characteristic patterns of vascular stenosis and neointima. Dissecting the mechanobiological signaling associated with local Eln depletion and subsequent smooth muscle cell response may help develop new therapeutic interventions for elastin-related diseases.