Smooth Muscle Cell Phenotypic Switching Research Articles

Dipyridamole and vascular healing following stent implantation.

Patients undergoing coronary stent implantation incur a 2% annual rate of adverse events, largely driven by in-stent restenosis (ISR) due to neointimal (NI) tissue proliferation, a process in which smooth muscle cell (SMC) biology may play a central role. Dipyridamole (DP) is an approved therapeutic agent with data supporting improved vascular patency rates. Pre-clinical data supports that DP may enact its vasculoprotective effects via adenosine receptor-A2B (ADOR-A2B). We sought to evaluate the efficacy of DP to mitigate ISR in a pre-clinical rabbit stent model. 24 New Zealand White Rabbits were divided into two cohorts-non-atherosclerosis and atherosclerosis (n = 12/cohort, 6 male and 6 female). Following stent implantation, rabbits were randomized 1:1 to control or oral dipyridamole therapy for 6 weeks followed by optical coherence tomography (OCT) and histology assessment of NI burden and stent strut healing. Compared to control, DP demonstrated a 16.6% relative reduction in NI volume (14.7 ± 0.8% vs. 12.5 ± 0.4%, p = 0.03) and a 36.2% relative increase in optimally healed stent struts (37.8 ± 2.8% vs. 54.6 ± 2.5%, p < 0.0001). Atherosclerosis demonstrated attenuated effect with no difference in NI burden (15.2 ± 1.0% vs. 16.9 ± 0.8%, p = 0.22) and only a 14.2% relative increase in strut healing (68.3 ± 4.1% vs. 78.7 ± 2.5%, p = 0.02). DP treated rabbits had a 44.6% (p = 0.045) relative reduction in NI SMC content. In vitro assessment of DP and coronary artery SMCs yielded dose-dependent reduction in SMC migration and proliferation. Selective small molecule antagonism of ADOR-A2B abrogated the effects of DP on SMC proliferation. DP modulated SMC phenotypic switching with ADOR-A2B siRNA knockdown supporting its role in the observed effects. Dipyridamole reduces NI proliferation and improves stent healing in a preclinical model of stent implantation with conventional antiplatelets. Atherosclerosis attenuates the observed effect. Clinical trials of DP as an adjunctive agent may be warranted to evaluate for clinical efficacy in stent outcomes.

Abstract 010: Interplay Between Nox5 And Endoplasmic Reticulum Stress Influences Vascular Smooth Muscle Cell Phenotypic Switching In Human Hypertension

Vascular remodelling, a hallmark of hypertension, involves vascular smooth muscle cell phenotypic switching. Molecular mechanisms underlying this process are unclear, but redox-dependent processes are important. Here we explored the role of Nox5, oxidative and ER stress in VSMC de-differentiation in hypertension. VSMCs from resistance arteries of normotensive (NT) and hypertensive (HT) subjects were studied. Nox5 compartmentalization (cell fractionation); ROS generation (chemiluminescence); activation of ER stress (IRE1α, PERK phosphorylation), and markers of phenotypic switching (αSMA, SM22, MYOCD KLF4) proliferation (PCNA) and inflammation/fibrosis (IL-6 and IL-8) were assessed. Nox5 and IRE1 were silenced with siRNA. In HT, Nox5 upregulation was compartment-specific: Nox5 levels were increased in the ER fraction (46 % vs NT, p&lt;0.05). IRE1α (95%) and PERK arms (36%) of ER stress were activated in HT (vs NT, p&lt;0.05). ROS scavenging (Tempol, 1mM) and Nox5 silencing reduced ER stress activation in HT (vs Ctl, p&lt;0.05). ER stress (4-PBA, 1mM) and IRE1 inhibition (STF083010, 60μM) reduced ROS levels and Nox5 expression in HT subjects (vs Ctl, p&lt;0.05). Induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression (25% vs Ctl) in NT (p&lt;0.05), suggesting association between Nox5-ROS and ER stress. Subsequently, we examined the impact of Nox5 and ER stress on VSMC phenotypic switching in HT. VSMCs from HT exhibited reduced expression of the VSMC markers αSMA (90%), SM22 (83%), and MYOCD (50%), whereas KLF4 levels (240%) were augmented (vs NT, p&lt;0.05). Additionally, expression of the proliferation marker, PCNA (217%) and pro-collagen I (23±2 vs NT:13±0.3ng/ml), and production of pro-inflammatory cytokines IL-6 (501±23 vs NT:121±6pg/mL) and IL-8 (373±34 vs NT:262±24pg/mL) were increased in HT (vs NT, p&lt;0.05). Nox5 and IRE1 silencing in VSMC from HT reduced PCNA expression, pro-collagen I release, and baseline and LPS-induced IL-6 and IL-8 release (vs Ctl, p&lt;0.05). Our findings indicate a circuitous relationship between Nox5 and ER stress where Nox5 is both upstream and downstream of ER stress. Activation of this system is closely linked to VSMC phenotypic switching, a crucial process in vascular dysfunction associated with hypertension.

Musashi-2 binds with Fbxo6 to induce Rnaset2 ubiquitination and chemokine signaling pathway during vascular smooth muscle cell phenotypic switch in atherosclerosis

ObjectiveThe objective of this study is to determine how Musashi-2 (MSI2) affects vascular smooth muscle cell (VSMC) phenotypic switch and contributes to atherosclerosis (AS). MethodsPrimary mouse VSMCs were transfected with MSI2 specific siRNA and treated with platelet-derived growth factor-BB (PDGF-BB). The proliferation, cell-cycle, and migration of VSMCs were determined by CCK-8, flow cytometry, wound healing, and transwell assays. Western blot and qRT-PCR were conducted to analyze the protein and mRNA expression. Moreover, the correlation between MSI2, Fbxo6, Rnaset2, and chemokine signaling was predicted and verified using RNAct database, KEGG, wiki, RNA-binding protein immunoprecipitation and co-immunoprecipitation. Moreover, H&E and Oil Red O staining were employed for assessing necrotic core and lipid accumulation in AS mouse aorta tissues. The numbers of B lymphocytes and monocytes, and the levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDL-C) in AS mice blood were investigated using flow cytometry and corresponding commercial kits, respectively. ResultsMSI2 was up-regulated in the PDGF-BB-treated VSMCs. Knockdown of MSI2 inhibited VSMC proliferation, cell-cycle, and migration. Moreover, MSI2 regulated VSMC phenotypic switch through binding with Fbxo6 to induce Rnaset2 ubiquitination. MSI2 knockdown inhibited chemokine signaling via regulating Fbxo6/Rnaset2 axis. In AS mice, knockdown of MSI2 inhibited the formation of necrotic core and atherosclerotic plaque, and inhibited chemokine signaling via regulating Fbxo6/Rnaset2 axis. ConclusionOur findings demonstrated that MSI2 could bind with Fbxo6 to induce Rnaset2 ubiquitination and the activation of chemokine signaling pathway during VSMC phenotypic switch in AS.

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.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.

Network Preservation Analysis Reveals Dysregulated Metabolic Pathways in Human Vascular Smooth Muscle Cell Phenotypic Switching.

Vascular smooth muscle cells are key players involved in atherosclerosis, the underlying cause of coronary artery disease. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. An in-depth characterization of their gene regulatory networks can help better understand how their dysfunction may impact disease progression. We conducted a gene expression network preservation analysis in aortic smooth muscle cells isolated from 151 multiethnic heart transplant donors cultured under quiescent or proliferative conditions. We identified 86 groups of coexpressed genes (modules) across the 2 conditions and focused on the 18 modules that are least preserved between the phenotypic conditions. Three of these modules were significantly enriched for genes belonging to proliferation, migration, cell adhesion, and cell differentiation pathways, characteristic of phenotypically modulated proliferative vascular smooth muscle cells. The majority of the modules, however, were enriched for metabolic pathways consisting of both nitrogen-related and glycolysis-related processes. Therefore, we explored correlations between nitrogen metabolism-related genes and coronary artery disease-associated genes and found significant correlations, suggesting the involvement of the nitrogen metabolism pathway in coronary artery disease pathogenesis. We also created gene regulatory networks enriched for genes in glycolysis and predicted key regulatory genes driving glycolysis dysregulation. Our work suggests that dysregulation of vascular smooth muscle cell metabolism participates in phenotypic transitioning, which may contribute to disease progression, and suggests that AMT (aminomethyltransferase) and MPI (mannose phosphate isomerase) may play an important role in regulating nitrogen and glycolysis-related metabolism in smooth muscle cells.

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.

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.

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.

SGC agonist BAY1021189 promotes thoracic aortic dissection formation by accelerating vascular smooth muscle cell phenotype switch

Thoracic aortic dissection (TAD) is common but lethal cardiovascular disease with high mortality. This study aimed to expound whether and how sGC-PRKG1 signaling pathway might promote the formation of TAD. Our work identified two modules with high relevance to TAD using WGCNA method. Combined with previous studies, we focused on the participation of endothelial NOS (eNOS) in the progression of TAD. Through immunohistochemistry, immunofluorescence and western blot we verified that eNOS expression was elevated in the tissues of patients and mice with aortic dissection, and the phosphorylation Ser1177 of eNOS was activated. In a BAPN-induced TAD mouse model, sGC-PRKG1 signaling pathway promotes TAD formation by inducing vascular smooth muscle cells (VSMCs) phenotype transition, which was demonstrated as a decrease in markers of the contractile phenotype of VSMCs such as αSMA, SM22α, and Calponin. These results were also verified by experiments in vitro. To explore the further mechanism, we conducted immunohistochemistry, western blot and quantitative RT-PCR (qPCR), the results of which indicated that sGC-PRKG1 signaling pathway was activated when TAD occurred. In conclusion, our current study revealed that sGC-PRKG1 signaling pathway could promote TAD formation by accelerating VSMCs phenotype switch.

Bestrophin3 Deficiency in Vascular Smooth Muscle Cells Activates MEKK2/3-MAPK Signaling to Trigger Spontaneous Aortic Dissection.

Aortic dissection (AD) is a fatal cardiovascular disorder without effective medications due to unclear pathogenic mechanisms. Bestrophin3 (Best3), the predominant isoform of bestrophin family in vessels, has emerged as critical for vascular pathological processes. However, the contribution of Best3 to vascular diseases remains elusive. Smooth muscle cell-specific and endothelial cell-specific Best3 knockout mice (Best3SMKO and Best3ECKO, respectively) were engineered to investigate the role of Best3 in vascular pathophysiology. Functional studies, single-cell RNA sequencing, proteomics analysis, and coimmunoprecipitation coupled with mass spectrometry were performed to evaluate the function of Best3 in vessels. Best3 expression in aortas of human AD samples and mouse AD models was decreased. Best3SMKO but not Best3ECKO mice spontaneously developed AD with age, and the incidence reached 48% at 72 weeks of age. Reanalysis of single-cell transcriptome data revealed that reduction of fibromyocytes, a fibroblast-like smooth muscle cell cluster, was a typical feature of human ascending AD and aneurysm. Consistently, Best3 deficiency in smooth muscle cells decreased the number of fibromyocytes. Mechanistically, Best3 interacted with both MEKK2 and MEKK3, and this interaction inhibited phosphorylation of MEKK2 at serine153 and MEKK3 at serine61. Best3 deficiency induced phosphorylation-dependent inhibition of ubiquitination and protein turnover of MEKK2/3, thereby activating the downstream mitogen-activated protein kinase signaling cascade. Furthermore, restoration of Best3 or inhibition of MEKK2/3 prevented AD progression in angiotensin II-infused Best3SMKO and ApoE-/- mice. These findings unveil a critical role of Best3 in regulating smooth muscle cell phenotypic switch and aortic structural integrity through controlling MEKK2/3 degradation. Best3-MEKK2/3 signaling represents a novel therapeutic target for AD.

Protein arginine methyltransferase 5-mediated arginine methylation stabilizes Kruppel-like factor 4 to accelerate neointimal formation.

Accumulating evidence supports the indispensable role of protein arginine methyltransferase 5 (PRMT5) in the pathological progression of several human cancers. As an important enzyme-regulating protein methylation, how PRMT5 participates in vascular remodelling remains unknown. The aim of this study was to investigate the role and underlying mechanism of PRMT5 in neointimal formation and to evaluate its potential as an effective therapeutic target for the condition. Aberrant PRMT5 overexpression was positively correlated with clinical carotid arterial stenosis. Vascular smooth muscle cell (SMC)-specific PRMT5 knockout inhibited intimal hyperplasia with an enhanced expression of contractile markers in mice. Conversely, PRMT5 overexpression inhibited SMC contractile markers and promoted intimal hyperplasia. Furthermore, we showed that PRMT5 promoted SMC phenotypic switching by stabilizing Kruppel-like factor 4 (KLF4). Mechanistically, PRMT5-mediated KLF4 methylation inhibited ubiquitin-dependent proteolysis of KLF4, leading to a disruption of myocardin (MYOCD)-serum response factor (SRF) interaction and MYOCD-SRF-mediated the transcription of SMC contractile markers. Our data demonstrated that PRMT5 critically mediated vascular remodelling by promoting KLF4-mediated SMC phenotypic conversion and consequently the progression of intimal hyperplasia. Therefore, PRMT5 may represent a potential therapeutic target for intimal hyperplasia-associated vascular diseases.

O015 CD34+ vascular stem cells play a key role in vein graft remodelling

Abstract Introduction Venous conduits that are grafted into arterial circulation undergo neointima remodelling, causing excessive hyperplasia that contributes to early graft failure which a major drawback of surgical vein grafting. Stem cells expressing the CD34 surface protein have been postulated to have a key role in the process, but their involvement has not been studied. Methods A segment of the inferior vena cava of wild-type mice was interposition grafted into mice in which CD34-expressing cells were located through their genetically induced expression of tdTomato red marker protein (CD34-CreERT2xRosa26-tdTomato mice). This enabled us to determine whether CD34+ derived cells found in the vein after grafting originated from the donor mouse or from the host. Grafts were explanted 4wks after surgery and analysed by immunohistology and single cell RNA sequencing to resolve cell-level gene expression. Cells from vein grafts were also cultured in vitro and proliferation/differentiation assays were carried out on purified CD34+ cells. Results 70% of cells in the vein graft neointima were derived from CD34+ graft lineage whereas there was no significant contribution of recipient-derived stem cells or bone marrow cells to the neointima. Vein graft neointima smooth muscle cells (SMC) are not stem cells derived. CD34+ stem cells differentiated predominantly into fibroblasts which induced vein SMC phenotype switching and proliferation. Conclusion The majority of CD34+ stem cells found in the neointima of veins, grafted into the arterial circulation were derived from the resident cell population. These cells

Calcium promotes vascular smooth muscle cell phenotypic switching in Marfan syndrome

Fibrillin 1 (Fbn1) mutations cause Marfan syndrome (MFS), with aortic root dilatation, dissection, and rupture. Few studies reported the blood calcium and lipid profile of MFS, and the effect of vascular smooth muscle cell (VSMC) phenotypic switching on MFS aortic aneurysm is unclear. Here, we aimed to investigate the role of calcium-related VSMC phenotypic switching in MFS. We retrospectively collected MFS patients' clinical data, performed bioinformatics analysis to screen the enriched biological process in MFS patients and mice, and detected markers of VSMC phenotypic switching on Fbn1C1039G/+ mice and primary aortic vascular smooth muscle cells. We found that patients with MFS have elevated blood calcium levels and dyslipidemia. Furthermore, the calcium concentration levels were increased with age in MFS mice, accompanied by the promoted VSMC phenotypic switching, and SERCA2 contributed to maintaining the contractile phenotype of VSMCs. This study provides the first evidence that the increased calcium is associated with the promoted VSMC phenotype switching in MFS. SERCA may become a novel therapeutic target for suppressing aneurysm progression in MFS.

Loss of tissue inhibitor of proteinase 3 (TIMP3), but not TIMP4, worsens abdominal and thoracic aortic aneurysm

Background: Aorta exhibits regional heterogeneity (structural and mechanical), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. TIMP3 and TIMP4 have shown region-dependent functions in aortic pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. Methods and Results: TAA or AAA was induced in male or female mice lacking TIMP3 ( Timp3 -/- ), TIMP4 ( Timp4 -/- ) or wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females. Timp3 -/- mice exhibited a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA whereas inflammation increased in the media post-AAA, but in the adventitia post-TAA. Timp3 -/- mice showed impaired intimal barrier integrity ( in vivo) following AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in TAA versus AAA. Timp4 -/- mice responded similarly to TAA and AAA induction as the WT. In vitro, Timp3 knockdown in human aortic EC more severely compromised the EC monolayer permeability compared to Timp4 knockdown and control groups following elastase treatment. In human aorta specimens, TIMP3 showed a region-specific expression pattern in TAA versus AAA consistent with the impact of its loss in the impaired intimal barrier in AAA, and the adventitial region in TAA. Conclusions: TIMP3 plays a protective role in both AAA and TAA, whereas loss of TIMP4 has minimal impact on the severity of aneurysm formation. TIMP3 loss in the aneurysmal aorta could be responsible for the inflammation, adverse remodeling and aortic dilation in AAA and TAA, through different mechanisms. Support: CIHR, Heart and Stroke. CIHR, Heart and Stroke. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 496: Inhibition Of Tirna-gly-gcc Ameliorates Neointimal Formation Via Cbx3-mediated Vascular Smooth Muscle Cells Phenotypic Switching

Introduction: Vascular smooth muscle cells (VSMCs) are the main component of blood vessels and synthetic VSMCs play an essential role in vascular diseases. tRNA-derived fragments (tRFs) are a new class of non-coding RNAs involved in a variety of pathological processes, but their biological functions and mechanisms in VSMCs phenotype transition and vascular intimal hyperplasia are unclear. Hypothesis: We assessed the hypothesis that tRFs play important role in VSMCs phenotype transition, regulation of tRFs could ameliorates neointimal formation. Methods: Differential expression profiles of tRFs from different phenotype of VSMCs was acquired using small-RNA sequence. The expression of tiRNA-Gly-GCC was confirmed in VSMCs, vascular tissues and plasma from healthy and atherosclerosis using quantitative real-time PCR and RNA fluorescence in situ hybridization. Gain-and loss- of function were performed using mimics and inhibitors to investigate the role of tiRNA-Gly-GCC in VSMCs phenotypic transition and neointimal formation after vascular injury. Dual-luciferase reporter gene and rescue experiments were performed to demonstrate the relationship between tiRNA-Gly-GCC and CBX3. Results: tiRNA-Gly-GCC is upregulated in synthetic VSMCs, atherosclerotic arteries, plasma and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses VSMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA-Gly- GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on VSMCs via downregulating the expression of CBX3. Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury in vivo . Conclusions: In conclusion, tiRNA-Gly-GCC is a mediator of VSMCs phenotypic switching by targeting CBX3, inhibition of tiRNA-Gly-GCC suppresses neointimal formation.

Abstract 701: Atherosclerosis Is A Smooth Muscle Cell-driven Tumor-like Disease

Background: Atherosclerosis, the leading cause of cardiovascular disease (CVD), is a chronic inflammatory disease involving pathological activation of multiple cell types, such as immunocytes (e.g., macrophage, T cells), smooth muscle cells (SMCs), endothelial cells, etc. Multiple lines of evidence have suggested that SMC “phenotypic switching” plays a central role in atherosclerosis development and complications. Yet, how SMCs and SMC-derived cells (SDCs) modulate atherosclerosis progression and clinical CVD remains poorly understood. Methods: We applied SMC lineage tracing mice and implemented comprehensive molecular and cellular biology, histological, computational, and pharmacological studies to reveal how tumor cell-like activities of SDCs drive the development of atherosclerosis. Results: Oxidative DNA damage occurs during SMC phenotypic switching and increases with the progression of atherosclerosis. Extensive DNA damage-induced genomic instability accumulates in both mouse and human atherosclerosis, especially in SDCs. Indeed, SDCs in atherosclerosis harbor multiple tumor cell-like features, including escape of replicative senescence, clonogenicity, invasive capacity, and ex vivo formation of three-dimensional (3D) spheroids like cancer stem cells. Multiple cancer-associated signaling pathways (e.g., p53, TNFa, NFkB, VEGF) are activated in SDCs. SMC-specific expression of oncogenic Kras G12D accelerates SMC phenotypic switching and exacerbates atherosclerosis. Moreover, we provide proof of concept in mouse models that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and regresses lesions in advanced disease. Conclusions: Our studies in human and mouse models provide the first systematic evidence that SDCs in atherosclerosis share extensive commonalities with tumor, which deepens our understanding of the disease and opens prospects for novel targeted strategies to prevent and treat atherosclerotic CVD.

Abstract 533: Doxycycline Fuels Mitochondrial Dysfunction In Aortic Smooth Muscle Cells

Introduction: The antibiotic doxycycline is known to inhibit inflammation, and was therefore considered as a therapeutic to prevent abdominal aortic aneurysm (AAA) growth. However, in two clinical trials, doxycycline-treated AAA patients did not experience reduced aneurysm growth. Doxycycline was previously shown to induce mitonuclear imbalance and reduce mitochondrial function. Objective: To determine that doxycycline would also impair mitochondrial function in the aorta and aortic smooth muscle cells (SMCs), giving rise to SMC phenotype switching. Results: Oral doxycycline intake induced mitonuclear imbalance in the murine aorta, human aortic aneurysm tissue and in human aortic SMCs. Consequently, doxycycline reduced mitochondrial activity and respiration in aortic SMCs. Transcription factor krüppel-like factor 4 (KLF4), a player in mitochondrial homeostasis, is upregulated after doxycycline treatment. Moreover, the integrated stress response effector, activating transcription factor 4 (ATF4), is enhanced to restore cellular homeostasis. These changes result in diminished expression of typical contractile SMC markers. Treatment with the drug Elamipretide (SS-31) improves mitochondrial markers and cellular stress management. Conclusion: Doxycycline leads to mitochondrial dysfunction and ER stress in aortic SMCs. These changes may contribute to aortic aneurysm growth by impairing SMC function. Elamipretide offers rescue from some of the harmful effects of doxycycline by improving mitochondrial function.

Homeobox A9 is a novel mediator of vascular smooth muscle cell phenotypic switching and proliferation by regulating methyl-CpG binding protein 2

Aberrant proliferation and phenotypic switching of vascular smooth muscle cells (VSMCs) are considered to be the main pathological processes of atherosclerotic plaque formation. Methyl-CpG binding protein 2 (MECP2) affects cell differentiation via modulating VSMC-specific gene expression and acts as a driver for the development of atherosclerosis (AS). Here, we aimed to elucidate the role of homeobox A9 (HOXA9) on aberrant VSMCs upon injury or AS, and whether HOXA9-mediated VSMC injury was associated with MECP2. Adeno-associated virus serotype 8-mediated knockdown of HOXA9 rescued aortic pathological injury of apolipoprotein E-deficient (ApoE−/−) mice fed a high-fat diet (HFD), characterized by the reduction of lipid accumulation and foam cell formation. Further in vitro evidence suggested that proliferation and migration of primary mouse VSMCs (mVSMCs) stimulated by oxidized low-density lipoprotein (ox-LDL) were inhibited after HOXA9 silencing. In addition, HOXA9 silencing blocked VSMC phenotypic switching from contractile to a pathological synthetic state. HOXA9 overexpression caused opposite alterations in ox-LDL-stimulated mVSMCs. Mechanistically, MECP2 was transcriptionally activated by HOXA9. Forced expression of MECP2 impaired the anti-proliferation, anti-migration, and phenotypic switching abilities of HOXA9 silencing in VSMCs upon ox-LDL stimulation. Collectively, our findings reveal that the role of HOXA9 in pathological vascular remodeling may attribute to transcriptional regulation of MECP2.

METTL14/YTHDF1 axis-modified UCHL5 aggravates atherosclerosis by activating the NLRP3 inflammasome

BackgroundVascular smooth muscle cell (VSMC) phenotypic switching contributes to VSMC proliferation and migration in atherosclerosis (AS). Nevertheless, the regulatory mechanism of VSMC phenotypic switching during AS progression is unclear. Here, the role and regulatory mechanism of UCHL5 in VSMC phenotypic switching during AS progression were investigated. MethodsApoE−/− mice were fed with high fat diet to establish AS model in vivo. VSMCs stimulated by ox-LDL were used as AS cellular model. VSMC proliferation and migration were examined by CCK8 assay and transwell assay, respectively. The levels of pro-inflammatory cytokines were assessed using ELISA. The interactions between METTL14/YTHDF1, UCHL5 and NLRP3 were analyzed using RIP and/or dual-luciferase reporter gene and/or Co-IP assays. NLRP3 ubiquitination was analyzed by ubiquitination analysis. ResultsUCHL5 was significantly upregulated in AS patients and ox-LDL-treated VSMCs. UCHL5 silencing ameliorated plaque formation and vascular remodeling in vivo and suppressed ox-LDL-induced VSMC proliferation, migration, inflammation and phenotypic switching in vitro. Moreover, METTL14 could increase UCHL5 mRNA m6A level and promoted UCHL5 expression by recruiting YTHDF1. Moreover, UCHL5 overexpression enhanced protein stability by deubiquitinating NLRP3. Rescue studies revealed that NLRP3 overexpression abrogated UCHL5 silencing-mediated biological effects in ox-LDL-treated VSMCs. ConclusionUCHL5 modified by METTL14/YTHDF1 axis could facilitate the inflammation and vascular remodeling in atherosclerosis by activating the NLRP3 inflammasome.