Neointima Formation Research Articles

Protective role of the nitric oxide synthases system in inter-organ communication

The role of the nitric oxide synthases (NOSs) system in inter-organ communication is not fully understood. We addressed this point in our mice lacking all three NOS isoforms (triple n/i/eNOSs-/- mice). We previously reported that the triple n/i/eNOSs-/- mice spontaneously develop myocardial infarction. However, it takes a long time (approximately 1 year) to develop myocardial infarction. We then revealed that 2/3-nephrectomized triple n/i/eNOSs-/- mice suddenly die due to early onset of myocardial infarction, succeeding in developing an experimentally useful model of myocardial infarction. These results suggest the protective role of NOSs in reno-cardiac communication. We next studied the role of NOSs in bone marrow cells (BM) in vascular lesion formation. Constrictive vascular remodeling and neointimal formation at 14 days after carotid artery ligation were markedly accelerated in wild-type (WT) mice transplanted with triple n/i/eNOSs-/- BM as compared with those with WT BM. These results suggest the protective role of NOSs in BM-vascular communication. We then investigated the role of NOSs in BM in pulmonary hypertension (PH). The extents of PH at 3 weeks after hypoxic exposure were markedly exacerbated in WT mice transplanted with triple n/i/eNOSs-/- BM as compared with those with WT BM, suggesting the protective role of NOSs in BM-lung communication. These lines of evidence indicate that systemic and BM NOSs may be a novel therapeutic target in MI, vascular disease, and PH.

Endothelial RIPK1 protects artery bypass graft against arteriosclerosis by regulating SMC growth.

RIPK1 is crucial in the inflammatory response. The process of vascular graft remodeling is also involved in endothelial inflammation, which can influence the behavior of smooth muscle cells. However, the role of endothelial RIPK1 in arterial bypass grafts remains unknown. Here, we established an arterial isograft mouse model in wild-type and endothelial RIPK1 conditional knockout mice. Progressive vascular remodeling and neointima formation occurred in the graft artery, showing SMC accumulation together with endothelial inflammatory adhesion molecule and cytokine expression. Endothelial RIPK1 knockout exacerbated graft stenosis by increasing secretion of N-Shh. Mechanistically, RIPK1 directly phosphorylated EEF1AKMT3 at Ser26, inhibiting its methyltransferase activity and global protein synthesis, which further attenuated N-Shh translation and secretion. Consistently, treatment with the Hedgehog pathway inhibitor GDC0449 markedly alleviated RIPK1 knockout-induced graft stenosis. Our results demonstrated that endothelial RIPK1 played a protective role in arterial bypass graft vascular remodeling, highlighting that targeting Hedgehog pathway may be an attractive strategy for graft failure in the future.

Omentin-1 enhances the inhibitory effect of endothelial progenitor cells on neointimal hyperplasia by inhibiting the p38 MAPK/CREB pathway

AimsEndothelial progenitor cells (EPCs) play an important role in vascular repair. However, they are dysfunctional in the inflammatory microenvironment during restenosis. In this study, we investigated whether omentin-1, an anti-inflammatory factor, could reduce neointima formation after carotid artery injury (CAI) in rats by improving EPC functions that were damaged by inflammation and the underlying mechanisms. Main methodsEPCs were transfected with adenoviral vectors expressing human omentin-1 or green fluorescent protein (GFP). Then, the rats received 2 × 106 EPCs expressing omentin-1 or GFP by tail vein injection directly after CAI and again 24 h later. Hematoxylin-eosin staining and immunohistochemistry were used for analyzing neointimal hyperplasia. Besides, EPCs were treated with omentin-1 and TNF-α to examine the underlying mechanism. Key findingsOur results showed that omentin-1 could significantly improve EPC functions, including proliferation, apoptosis and tube formation. Meanwhile, EPCs overexpressed with omentin-1 could significantly reduce neointimal hyperplasia and tumor necrosis factor-α (TNF-α) expression after CAI in rats. TNF-α could notably induce EPC dysfunction, which could be markedly reversed by omentin-1 through the inhibition of the p38 MAPK/CREB pathway. Furthermore, a p38 MAPK agonist (anisomycin) significantly abrogated the protective effects of omentin-1 on EPCs damaged by TNF-α. SignificanceOur results indicated that genetically modifying EPC with omentin-1 could be an alternative strategy for the treatment of restenosis.

SMYD2 regulates vascular smooth muscle cell phenotypic switching and intimal hyperplasia via interaction with myocardin.

The SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase that has been reported to regulate carcinogenesis and inflammation. However, its role in vascular smooth muscle cell (VSMC) homeostasis and vascular diseases has not been determined. Here, we investigated the role of SMYD2 in VSMC phenotypic modulation and vascular intimal hyperplasia and elucidated the underlying mechanism. We observed that SMYD2 expression was downregulated in injured carotid arteries in mice and phenotypically modulated VSMCs in vitro. Using an SMC-specific SMYD2 knockout mouse model, we found that SMYD2 ablation in VSMCs exacerbated neointima formation after vascular injury in vivo. Conversely, SMYD2 overexpression inhibited VSMC proliferation and migration in vitro and attenuated arterial narrowing in injured vessels in mice. SMYD2 downregulation promoted VSMC phenotypic switching accompanied with enhanced proliferation and migration. Mechanistically, genome-wide transcriptome analysis and loss/gain-of-function studies revealed that SMYD2 up-regulated VSMC contractile gene expression and suppressed VSMC proliferation and migration, in part, by promoting expression and transactivation of the master transcription cofactor myocardin. In addition, myocardin directly interacted with SMYD2, thereby facilitating SMYD2 recruitment to the CArG regions of SMC contractile gene promoters and leading to an open chromatin status around SMC contractile gene promoters via SMYD2-mediated H3K4 methylation. Hence, we conclude that SMYD2 is a novel regulator of VSMC contractile phenotype and intimal hyperplasia via a myocardin-dependent epigenetic regulatory mechanism.

Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization.

Cell migration is essential for many biological and pathological processes. Establishing cell polarity with a trailing edge and forming a single lamellipodium at the leading edge of the cell is crucial for efficient directional cell migration and is a hallmark of mesenchymal cell motility. Lamellipodia formation is regulated by spatial-temporal activation of the small GTPases Rac and Cdc42at the front edge, and RhoA at the rear end. At a molecular level, partitioning-defective (Par) protein complex comprising Par3, Par6, and atypical Protein Kinase (aPKC isoforms ζ and λ/ι) regulates front-rear axis polarization. At the front edge, integrin clustering activates Cdc42, prompting the formation of Par3/Par6/aPKC complexes to modulate MTOC positioning and microtubule stabilization. Consequently, the Par3/Par6/aPKC complex recruits Rac1-GEF Tiam to activate Rac1, leading to lamellipodium formation. At the rear end, RhoA-ROCK phosphorylates Par3 disrupting its interaction with Tiam and inactivating Rac1. RhoA activity at the rear end allows the formation of focal adhesions and stress fibers necessary to generate the traction forces that allow cell movement. Nox1-based NADPH oxidase is necessary for PDGF-induced migration in vitro and in vivo for many cell types, including fibroblasts and smooth muscle cells. Here, we report that Nox1-deficient cells failed to acquire a normal front-to-rear polarity, polarize MTOC, and form a single lamellipodium. Instead, these cells form multiple protrusions that accumulate Par3 and active Tiam. The exogenous addition of H2O2 rescues this phenotype and is associated with the hyperactivation of Par3, Tiam, and Rac1. Mechanistically, Nox1 deficiency induces the inactivation of PP2A phosphatase, leading to increased activation of aPKC. These results were validated in Nox1y/- primary mouse aortic smooth muscle cells (MASMCs), which also showed PP2A inactivation after PDGF-BB stimulation consistent with exacerbated activation of aPKC. Moreover, we evaluated the physiological relevance of this signaling pathway using a femoral artery wire injury model to generate neointimal hyperplasia. Nox1y/- mice showed increased staining for the inactive form of PP2A and increased signal for active aPKC, suggesting that PP2A and aPKC activities might contribute to reducing neointima formation observed in the arteries of Nox1y/- mice.

The roles of METTL3 on autophagy and proliferation of vascular smooth muscle cells are mediated by mTOR rather than by CDK1

BackgroundAberrant proliferation of vascular smooth muscle cells (VSMCs) is the cause of neointima formation followed by vascular injury. Autophagy is involved in this pathological process, but its function is controversial. Recently, we found that methyltransferase like 3 (METTL3) inhibited VSMC proliferation by activating autophagosome formation. Moreover, we also demonstrated that METTL3 reduced the levels of phosphorylated mammalian target of rapamycin (p-mTOR) and cyclin dependent kinase 1 (p-CDK1/CDC2), which were critical for autophagy and proliferation regulation. However, whether mTOR and CDK1 mediated the function of METTL3 on autophagy and proliferation in VSMCs remains unknown.ResultsWe showed that the activator of mTOR, MHY1485 largely reversed the effects of METTL3 overexpression on VSMC autophagy and proliferation. Rapamycin, the inhibitor of mTOR, obviously nullified the pro-proliferation effects of METTL3 knockdown by activating autophagy in VSMCs. Unexpectedly, mTOR did not contribute to the impacts of METTL3 on migration and phenotypic switching of VSMCs. On the other hand, by knockdown of CDK1 in VSMC with METTL3 deficiency, we demonstrated that CDK1 was involved in METTL3-regulated proliferation of VSMCs, but this effect was not mediated by autophagy.ConclusionsWe concluded that mTOR but not CDK1 mediated the role of METTL3 on VSMC proliferation and autophagy.

P4HA2-induced prolyl hydroxylation of YAP1 restricts vascular smooth muscle cell proliferation and neointima formation

Vascular smooth muscle cell (VSMC) proliferation and neointima formation play significant roles in atherosclerosis development and restenosis following percutaneous coronary intervention. Our team previously discovered that TEA domain transcription factor 1 (TEAD1) promotes vascular smooth muscle differentiation, which is necessary for vascular development. Conversely, aberrant YAP1 activation upregulates the platelet-derived growth factor receptor beta to encourage VSMC proliferation and neointima formation. In this study, we aimed to investigate the molecular mechanisms of YAP1/TEAD signaling during neointima formation. Our research focused on the prolyl 4-hydroxylase alpha 2 (P4HA2) and its downstream target, Yes-associated protein 1 (YAP1), in regulating VSMC differentiation and neointima formation. Our results indicated that P4HA2 reduction leads to VSMC dedifferentiation and promotes neointima formation after injury. Furthermore, we found that P4HA2-induced prolyl hydroxylation of YAP1 restricts its transcriptional activity, which is essential to maintaining VSMC differentiation. These findings suggest that targeting P4HA2-mediated prolyl hydroxylation of YAP1 may be a promising therapeutic approach to prevent injury-induced neointima formation in cardiovascular disease.

Abstract P3045: MicroRNA-31-5p Is Upregulated During Neointima Formation And Promotes Smooth Muscle Cell Proliferation And Migration By Regulating Krüppel-like Factor 4 (KLF4)

In the context of CVDs, microRNAs (miRs) have emerged as potential therapeutic targets for preventing adverse vascular remodeling and restenosis, given their ability to modulate multiple signaling pathways and cellular processes. Here, we propose that targeting miR-31-5p might be an effective approach to enhance vascular healing and regeneration in a cell-specific manner, thereby limiting angioplasty-related complications. A screening of murine neointimal tissue for differentially regulated miRs revealed robustly increased expression levels of miR-31-5p over time. Subsequently, expression levels of miR-31-5p were confirmed by qRT-PCR in human coronary artery endothelial cells (EC) and -smooth muscle cells (SMC). Further, the effect of miR-31-5p on cellular function was investigated and possible targets were identified and confirmed on mRNA and protein level. Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in C57BL/6J mice following wire-induced injury of the femoral artery at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in SMC (p<0,05 ) following serum stimulation, whereas only a slightly enhanced expression was observed in EC. On a functional level, miR-31-5p revealed dissenting effects on EC and SMC: Migration and proliferation were not altered in EC (p>0.05). In contrast, knockdown of miR-31-5p significantly reduced the migration (p<0,05) and proliferation in SMC (p<0,01) without detectable apoptotic effects. Systematic in silico target screens suggested KLF4 as potential target. Knock down of miR-31-5p resulted in a significant upregulation of KLF4 in SMC (p<0,01), whereas overexpression resulted in a significant downregulation of KLF4 in SMC (p<0,05) on mRNA- and protein level. Here, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional role in SMC rather than EC since knockdown of miR-31-5p was able to prevent SMC proliferation and migration. Mechanistically, KLF4 identified as a direct target of miR-31-5p can mediate the observed effects. Thus, miR-31-5p might represent an attractive target to selectively modulate SMC dysfunction following vascular intervention, limiting neointima formation in treated vessels.

Flk1 Deficiency and Hypoxia Synergistically Promote Endothelial Dysfunction, Vascular Remodeling, and Pulmonary Hypertension.

The mechanisms underlying pulmonary hypertension (PH) remain largely unknown; further, why advanced vascular remodeling preferentially occurs in arterioles is yet to be answered. VEGF (vascular endothelial growth factor) regulates angiogenesis through Flk1 and Flt1 on endothelial cells (ECs), which may be related to PH pathogenesis. However, spatiotemporal expression patterns of Flk1 and Flt1 in the pulmonary vascular system and the role of endothelial Flk1 in PH development remain poorly understood. We analyzed multiple reporter mice, including Flk1-GFP bacterial artificial chromosome transgenic (Tg), Flt1-DsRed bacterial artificial chromosome Tg, and Flk1-GFP/Flt1-DsRed double Tg mice, to determine the spatiotemporal expression of Flk1 and Flt1 in hypoxia-induced PH. We also used Cdh5CreERT2/Flk1f/f/Tomato (Flk1-KO) mice to induce EC-specific Flk1 deletion and lineage tracing in chronic hypoxia. Flk1 was specifically expressed in the ECs of small pulmonary vessels, including arterioles. Conversely, Flt1 was more broadly expressed in the ECs of large- to small-sized vessels in adult mouse lungs. Intriguingly, Flk1+ ECs were transiently increased in hypoxia with proliferation, whereas Flt1 expression was unchanged. Flk1-KO mice did not exhibit pulmonary vascular remodeling nor PH in normoxia; however, the arteriolar ECs changed to a cuboidal shape with protrusion. In hypoxia, Flk1 deletion exacerbated EC dysfunction and reduced their number via apoptosis. Additionally, Flk1 deletion promoted medial thickening and neointimal formation in arterioles and worsened PH. Mechanistically, lineage tracing revealed that neointimal cells were derived from Flk1-KO ECs. Moreover, RNA sequencing in pulmonary ECs demonstrated that Flk1 deletion and hypoxia synergistically activated multiple pathways, including cell cycle, senescence/apoptosis, and cytokine/growth factor, concomitant with suppression of cell adhesion and angiogenesis, to promote vascular remodeling. Flk1 and Flt1 were differentially expressed in pulmonary ECs. Flk1 deficiency and hypoxia jointly dysregulated arteriolar ECs to promote vascular remodeling. Thus, dysfunction of Flk1+ ECs may contribute to the pathogenesis of advanced vascular remodeling in pulmonary arterioles.

Topographic distribution of inflammation factors in a healing aneurysm

BackgroundHealing 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.MethodsDecellularized 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.ResultsCoiled 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.ConclusionThe 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.

Large, Wide-Neck, Side-Wall Aneurysm Treatment in Canines Using NeuroCURE: A Novel Liquid Embolic.

Untreated intracranial aneurysms can rupture and result in high rates of morbidity and mortality. Although there are numerous approved endovascular aneurysm treatment devices, most require dual anti-platelet therapy, are minimally biocompatible, or are prone to recanalization. Neurovascular Controlled Uniform Rapid Embolic (NeuroCURE) is an innovative polymer gel material with long-term stability, biocompatibility, and hemocompatibility developed for the treatment of large, wide-neck aneurysms. Sidewall aneurysms were surgically created in 10 canines and NeuroCURE was injected through a 0.025 microcatheter under a single balloon inflation period. Aneurysm treatment was angiographically assessed post-embolization and pre-term with Raymond-Roy occlusion classification and a qualitative flow grade scale. Aneurysm neck stability and biocompatibility was histologically assessed to grade platelet/fibrin thrombus, percent endothelialization, and neointimal formation. Aneurysm sac stability was assessed by NeuroCURE sac content, inflammation, and neo-angiogenesis scales. Explanted aneurysms exhibited a smooth surface at the aneurysm neck with nearly complete neointimal coverage at 3-months. By 6-months, neck endothelialization was 100% in all animals (average Raymond-Roy occlusion classification of 1.2), with no instances of aneurysm recanalization or parent vessel flow compromise. Biocompatibility assessments verified a lack of inflammatory response, neo-angiogenesis, and platelet/fibrin thrombus formation. The NeuroCURE material promotes progressive occlusion of wide-necked side wall aneurysms over time without the need for dual antiplatelet agents. NeuroCURE also promotes neointimal tissue infill without dependence on thrombus formation and thus resists aneurysm recanalization. NeuroCURE remains a compelling investigational device for the treatment of intracranial aneurysms.

The innate immune sensor STING accelerates neointima formation via NF-κB signaling pathway.

Vascular smooth muscle cells (VSMCs) proliferation, migration, and phenotypic switching are considered crucial events in the progression of neointima formation. Stimulator of interferon genes (STING), an innate immune sensor of cyclic dinucleotides against pathogens, in neointima formation remains obscure. Here, we observed a significant increase in STING expression on the neointima of injured vessels and mouse aortic VSMCs induced by PDGF-BB. In vivo, global knockout of STING (Sting-/-) attenuated neointima formation after vascular injury. In vitro data showed that STING deficiency significantly alleviated PDGF-BB-induced proliferation and migration in VSMCs. Furthermore, these contractile marker genes were upregulated in Sting-/- VSMCs. Overexpression of STING promoted proliferation, migration, and phenotypic switching in VSMCs. Mechanistically, STING-NF-κB signaling was involved in this process. The pharmacological inhibition of STING induced by C-176 partially prevented neointima formation due to suppression of VSMCs proliferation. Taken together, STING-NF-κB axis significantly promoted proliferation, migration, and phenotypic switching of VSMCs, which may be a novel therapeutic approach to combat vascular proliferative diseases.

Three-dimensional reconstruction imaging by C-arm computed tomography accurately visualizes in-stent neointimal formation in patients with stent-assisted coil embolization.

Stent apposition to the vessel wall and in-stent neointimal formation after stent-assisted coil embolization for intracranial aneurysm are important factors associated with postoperative thromboembolic complications. No assessment methods have been established to depict 3-dimensional (3D) all-round in-stent neointimal formation. To demonstrate the superiority of Dyna-3D imaging assessment as a modality for all-round ISNF in comparison with conventional two-dimensional digital subtraction angiography (2D-DSA). Consecutive patients who underwent braided stent-assisted coil embolization for unruptured aneurysm between November 2016 and September 2021 were enrolled. Radiological assessments for stent apposition to the parent vessel after stent deployment and in-stent neointimal formation after 3 months were obtained. Dyna-3D was reconstructed by overlapping a plain image showing stent struts with a rotational DSA image showing the vessel lumen. Reconstructed Dyna-3D images can be rotated to any angle on the screen to evaluate to stent apposition around the vessel and in-stent neointimal formation in 3D, for comparison with 2D-DSA evaluations. Among the 73 patients enrolled, 70 patients (96%) showed complete stent wall apposition on Dyna-3D. Higher intra-rater agreement was confirmed on assessment of in-stent neointimal formation with Dyna-3D (Cohen's κ = 0.811) than with conventional 2D-DSA (Cohen's κ = 0.517). in-stent neointimal formation could not be confirmed on conventional imaging in 9 cases (16%) and on Dyna-3D in 2 cases (3%). The number of in-stent neointimal formations rated as stent wire completely outside the endothelial line was significantly higher with Dyna-3D than with 2D-DSA (p = 0.0001). All-round 3D evaluation by Dyna-3D imaging appears useful for confirming in-stent neointimal formation after braided stent deployment in patients after stent-assisted coil embolization.

Epigenetic regulation of vascular smooth muscle cell phenotypic switch and neointimal formation by PRMT5.

Phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic state is involved in the development of cardiovascular diseases, including atherosclerosis, hypertension, and post-angioplasty restenosis. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has been implicated in multiple cellular processes, however, its role in VSMC biology remains undetermined. The objective of this study was to determine the role of PRMTs in VSMC phenotypic switch and vascular remodelling after injury. Our results show that PRMT5 is the most abundantly expressed PRMT in human aortic SMCs, and its expression is up-regulated in platelet-derived growth factor (PDGF)-stimulated VSMCs, human atherosclerotic lesions, and rat carotid arteries after injury, as determined by western blot and immunohistochemical staining. PRMT5 overexpression inhibits the expression of SMC marker genes and promotes VSMC proliferation and migration, while silencing PRMT5 exerts the opposite effects. Mechanistically, we found that PRMT5 overexpression led to histone di-methylation of H3R8 and H4R3, which in turn attenuates acetylation of H3K9 and H4, thus limiting recruitment of the SRF/myocardin complexes to the CArG boxes of SMC marker genes. Furthermore, both SMC-specific deletion of PRMT5 in mice and local delivery of lentivirus expressing shPRMT5 to rat carotid arteries significantly attenuated neointimal formation after injury. Likewise, pharmacological inhibition of PRMT5 by EPZ015666 markedly inhibited carotid artery ligation-induced neointimal formation in mice. Our results identify PRMT5 as a novel regulator in VSMC phenotypic switch and suggest that inhibition of PRMT5 may represent an effective therapeutic strategy for proliferative vascular diseases.

Multilevel Assessment of Stent-Induced Inflammation in the Adjacent Vascular Tissue.

A recent U.S. Food and Drug Administration report presented the currently available scientific information related to biological response to metal implants. In this work, a multilevel approach was employed to assess the implant-induced and biocorrosion-related inflammation in the adjacent vascular tissue using a mouse stent implantation model. The implications of biocorrosion on peri-implant tissue were assessed at the macroscopic level via in vivo imaging and histomorphology. Elevated matrix metalloproteinase activity, colocalized with the site of implantation, and histological staining indicated that stent surface condition and implantation time affect the inflammatory response and subsequent formation and extent of neointima. Hematological measurements also demonstrated that accumulated metal particle contamination in blood samples from corroded-stetted mice causes a stronger immune response. At the cellular level, the stent-induced alterations in the nanostructure, cytoskeleton, and mechanical properties of circulating lymphocytes were investigated. It was found that cells from corroded-stented samples exhibited higher stiffness, in terms of Young's modulus values, compared to noncorroded and sham-stented samples. Nanomechanical modifications were also accompanied by cellular remodeling, through alterations in cell morphology and stress (F-actin) fiber characteristics. Our analysis indicates that surface wear and elevated metal particle contamination, prompted by corroded stents, may contribute to the inflammatory response and the multifactorial process of in-stent restenosis. The results also suggest that circulating lymphocytes could be a novel nanomechanical biomarker for peri-implant tissue inflammation and possibly the early stage of in-stent restenosis. Large-scale studies are warranted to further investigate these findings.

Autophagy protein 5 controls flow-dependent endothelial functions

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Epigenetic Reader Bromodomain-Containing Protein 4 in Aging-Related Vascular Pathologies and Diseases: Molecular Basis, Functional Relevance, and Clinical Potential.

Aging is a key independent risk factor of various vascular diseases, for which the regulatory mechanisms remain largely unknown. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain and Extra-Terminal domain (BET) family and is an epigenetic reader playing diverse roles in regulating transcriptional elongation, chromatin remodeling, DNA damage response, and alternative splicing in various cells and tissues. While BRD4 was initially recognized for its involvement in cancer progression, recent studies have revealed that the aberrant expression and impaired function of BRD4 were highly associated with aging-related vascular pathology, affecting multiple key biological processes in the vascular cells and tissues, providing new insights into the understanding of vascular pathophysiology and pathogenesis of vascular diseases. This review summarizes the recent advances in BRD4 biological function, and the progression of the studies related to BRD4 in aging-associated vascular pathologies and diseases, including atherosclerosis, aortic aneurism vascular neointima formation, pulmonary hypertension, and essential hypertension, providing updated information to advance our understanding of the epigenetic mechanisms in vascular diseases during aging and paving the way for future research and therapeutic approaches.

Conventional Platinum Metal Implants Provoke Restenosis Responses in Atherogenic but Not Healthy Arteries

Platinum-containing stents are commonly used in humans with hypercholesterolemia, whereas preclinical stent evaluation has commonly been performed in healthy animal models, providing inadequate information about stent performance under hypercholesterolemic conditions. In this investigation, we used an ApoE−/− mouse model to test the impact of hypercholesterolemia on neointima formation on platinum-containing implants. We implanted 125 μm diameter platinum wires into the abdominal aortas of ApoE−/− and ApoE+/+ mice for 6 months, followed by histological and immunofluorescence examination of neointimal size and composition. It was found that ApoE−/− mice developed neointimas with four times larger area and ten times greater thickness than ApoE+/+ counterparts. Neointimas developed in the ApoE−/− mice also contained higher amounts of lipids quantified as having 370 times more coverage compared to ApoE+/+, a 3-fold increase in SMCs, and a 22-fold increase in macrophages. A confluent endothelium had regenerated in both mouse strains. The ApoE−/− mice experienced luminal reductions more closely resembling clinically relevant restenosis in humans. Overall, the response to platinum arterial implants was highly dependent upon the atherogenic environment.

BRD4770 inhibits vascular smooth muscle cell proliferation via SUV39H2, but not EHMT2 to protect against neointima formation.

The behavior of vascular smooth muscle cells (VSMCs) contributes to the formation of neointima. We previously found that EHMT2 suppressed autophagy activation in VSMCs. BRD4770, an inhibitor of EHMT2/G9a, plays a critical role in several kinds of cancers. However, whetherand how BRD4770 regulates the behavior of VSMCs remain unknown. In this study, we evaluate the cellular effect of BRD4770 on VSMCs by series of experiments in vivo and ex vivo. We demonstrated that BRD4770 inhibited VSMCs' growth by blockage in G2/M phase in VSMCs. Moreover, our results demonstrated that the inhibition of proliferation was independent on autophagy or EHMT2 suppression which we previous reported. Mechanistically, BRD4770 exhibited an off-target effect from EHMT2 and our further study reveal that the proliferation inhibitory effect by BRD4770 was associated with suppressing on SUV39H2/KTM1B. In vivo, BRD4770 was also verified to rescue VIH. Thus, BRD4770 function as a crucial negative regulator of VSMC proliferation via SUV39H2 and G2/M cell cycle arrest and BRD4770 could be a molecule for the therapy of vascular restenosis.

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.