Vascular Smooth Muscle Cell Dedifferentiation Research Articles

Ginger protects against vein graft remodeling by precisely modulating ferroptotic stress in vascular smooth muscle cell dedifferentiation

Vein graft failure (VGF) is associated with vein graft (VG) intimal hyperplasia, which is characterized by abnormal accumulation of vascular smooth muscle cells (VSMCs). Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition, also known as dedifferentiation. There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedifferentiation, exerting vasoprotective functions; however, the precise mechanisms have not been fully characterized. Therefore, we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation. Ginger significantly inhibited neointimal hyperplasia and promoted lumen opening in a 3-month VG, which was primarily achieved by reducing ferroptotic stress. Ferroptotic stress is a pro-ferroptotic state. Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type, forming neointima after vein transplantation. Ginger and its two main vasoprotective ingredients (6-gingerol and 6-shogaol) inhibit VSMC dedifferentiation by reducing ferroptotic stress. Network pharmacology analysis revealed that 6-gingerol inhibits ferroptotic stress by targeting P53 while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase (Alox5), both promoting ferroptosis. Furthermore, both ingredients co-target peroxisome proliferator-activated receptor gamma (PPARγ), decreasing PPARγ mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) expression. Nox1 promotes intracellular reactive oxygen species (ROS) production and directly induces VSMC dedifferentiation. In addition, Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production, indirectly leading to VSMC dedifferentiation. Ginger, a natural multi-targeted ferroptotic stress inhibitor, finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells.

De-differentiation and subsequent increased proliferation and inflammation of vascular smooth muscle cells (VSMCs) is one of the mechanisms of atherogenesis. Maintaining VSMCs in a contractile differentiated state is therefore a promising therapeutic strategy for atherosclerosis. We have reported the 18-base myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes skeletal and myocardial differentiation. The present study investigated the effect of iSN04 on VSMCs because nucleolin has been reported to contribute to VSMC de-differentiation under pathophysiological conditions. Nucleolin is localized in the nucleoplasm and nucleoli of both rat and human VSMCs. iSN04 without a carrier was spontaneously incorporated into VSMCs, indicating that iSN04 would serve as an anti-nucleolin aptamer. iSN04 treatment decreased the ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive proliferating VSMCs and increased the expression of α-smooth muscle actin, a contractile marker of VSMCs. iSN04 also suppressed angiogenesis of mouse aortic rings ex vivo, which is a model of pathological angiogenesis involved in plaque formation, growth, and rupture. These results demonstrate that antagonizing nucleolin with iSN04 preserves VSMC differentiation, providing a nucleic acid drug candidate for the treatment of vascular disease.

The role of FGL2 in vascular smooth muscle cell dysfunction in coronary artery bypass vein graft failure

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation Rationale The sudden exposure of venous endothelial cells (vECs) to arterial fluid shear stress (FSS) is thought to be a major contributor to coronary artery bypass vein graft failure (VGF). However, the acute effects of arterial FSS on the vEC secretome and the corresponding effects on underlying vascular smooth muscle cells (VSMCs) remains poorly characterised. Methods & Results Human umbilical vein endothelial cells (HUVECs; n=6) pre-conditioned to venous FSS (18h; 1.5 dynes/cm2) were further exposed to venous or arterial FSS (24h; 15 dynes/cm2) using an Ibidi pump system. Analysis of the EC secretome using Tandem Mass Tagging proteomics detected significantly increased fibroleukin (FGL2) in conditioned media from ECs exposed to arterial FSS compared to venous FSS (fold-change 1.97±0.72; P=0.0078). In addition, RT-qPCR analysis of FGL2 production showed a significant increase in vECs exposed to arterial FSS (2.11±0.27 vs. 0.05±0.04 log(copy number/mg); P=0.0312). Subsequently, pooled human saphenous vein VSMCs (3 donors per pool) were treated with conditioned media from ECs exposed to arterial FSS (n=6) which showed a significant increase in apoptosis, measured by active cleaved caspase-3 (CC3) immunocytochemistry (27±1% vs 22±1%; P=0.0391), and a significant decrease in the contractile markers smoothelin and desmin quantified by Western blotting (fold-change 0.76±0.06 and 0.44±0.11; P=0.004 and P=0.0421, respectively), in comparison to VSMCs treated with conditioned media from ECs exposed to venous FSS. Treatment of VSMCs (n=6) with recombinant FGL2 (20 ng/mL) for 72h likewise resulted in increased apoptosis (27±4% vs. 16±3%; P=0.0169) and decreased desmin and smoothelin (fold-change 0.68±0.03 and 0.61±0.07; P=0.0322 and P=0.0479, respectively). Conclusions The exposure of vECs to arterial FSS results in increased production and release of FGL2 which induced VSMC apoptosis and de-differentiation and therefore potentially contributes to VGF. Consequently, targeting FGL2 production or release may provide a therapeutic target to improve vein graft patency.

Dynamin-related protein 1 mediates the therapeutic effect of isoliquiritigenin in diabetic intimal hyperplasia via regulation of mitochondrial fission.

Phenotypic shift of vascular smooth muscle cells (VSMCs) plays a key role in intimal hyperplasia, especially in patients with diabetes mellitus (DM). This study aimed to investigate the role of dynamin-related protein 1 (DRP1) in mitochondrial fission-mediated VSMC phenotypic shift and to clarify whether DRP1 is the therapeutic target of isoliquiritigenin (ISL). Wire injury of carotid artery or platelet-derived growth factor treatment was performed in DM mice or high-glucose cultured human aortic smooth muscle cells (HASMCs), respectively. The effects of DRP1 silencing on DM-induced intimal hyperplasia were investigated both in vivo and in vitro. Phenotypic shift of HASMCs was evaluated by detection of reactive oxygen species (ROS) generation, cell viability, and related protein expressions. The effects of ISL on DM-induced intimal hyperplasia were evaluated both in vivo and in vitro. DRP1 silencing and ISL treatment attenuated DM-induced intimal hyperplasia with reduced ROS generation, cell viability, and VSMC dedifferentiation. The GTPase domain of DRP1 protein played a critical role in mitochondrial fission in DM-induced VSMC phenotypic shift. Cellular experiments showed that ISL inhibited mitochondrial fission and reduced the GTPase activity of DRP1, which was achieved by the directly binding to K216 of the DRP1 GTPase domain. ISL attenuated mouse intimal hyperplasia by reducing GTPase activity of DRP1 and inhibiting mitochondrial fission in vivo. In conclusion, increased GTPase activity of DRP1 aggregated DM-induced intimal hyperplasia by increasing mitochondrial fission-mediated VSMC phenotypic shift. ISL attenuated mouse intimal hyperplasia by reducing DRP1 GTPase activity and inhibiting mitochondrial fission of VSMCs.

Abstract 3053: An Emerging Role Of The RNA Binding Protein Human Antigen R In Atherosclerotic Vascular Smooth Muscle Cell Heterogeneity

Objective: Lineage tracing studies have shown that advanced atherosclerosis causes vascular smooth muscle cells (VSMCs) to become macrophage-like (MLCs), which increases inflammation. We investigated whether the RNA binding protein human antigen R (HuR) and the inflammatory receptor protease activated receptor 2 (PAR2) accelerate VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Downregulation of VSMC migration and cytokine release in Par2 -/- mice reduces atherosclerotic lesion area compared to proficient controls. To examine the role of PAR2 in the VSMC-MLC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10μg/mL methyl-β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers and upregulated macrophage markers, Par2 -/- VSMCs remained stable. When comparing 101 mice strains with induced atherosclerosis, PAR2 was significantly correlated with Krüppel-like factor 4 (KLF4), a gene that regulates VSMC dedifferentiation. Also, Par2-/- VSMCs had lower basal KLF4 expression. Previous studies have demonstrated PAR2 mRNA is bound and stabilized by the RNA binding protein HuR. Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression, suggesting a role of HuR in PAR2 mRNA stability. Our studies have also demonstrated a dampening of HuR activation in Par2 -/- VSMCs, suggesting a potential feedforward mechanism between HuRs stabilizing capabilities of PAR2 mRNA and PAR2s presence necessary for HuR activation. To delineate the role of HuR in VSMC dedifferentiation and atherosclerosis, a HuR flox mouse line has been bred on to a SM22 Cre as well as a Myh11 Cre lineage tracing reporter line. To date, we have determined male and female Ldlr -/- HuR flox/flox SM22 Cre+ mice have attenuated atherosclerosis compared to their littermate Cre - controls at both 12 and 24 weeks. Conclusions: These results suggest that VSMC PAR2 activation mediates the dedifferentiation of VSMCs via KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. We also have established a definitive role of VSMC HuR in the development of atherosclerosis. Future studies will continue to elucidate the specific role of HuR in VSMC dedifferentiation.

Abstract 3114: Time Course Multi-omics Approach Investigating Gene Expression Regulation During Vascular Smooth Muscle Cell Phenotypic Transitioning

Coronary artery disease (CAD) is the leading cause of death in the United States. There is a need for novel therapeutic targets that prevent life-threatening consequences directly associated with plaque accumulation in the vascular wall—the underlying cause of CAD. During the progression of atherosclerosis, vascular smooth muscle cells (VSMCs) transdifferentiate from a contractile to a synthetic phenotype that can either promote fibrous cap stability (fibroblast-like) or contribute to inflammation and plaque accumulation (macrophage or osteoblast-like).The molecular mechanisms driving the functional phenotypic switching of VSMCs are not fully understood and represent a unique opportunity for discovering new treatments directly targeting plaque formation in the vascular wall. We measured gene expression regulation during VSMC dedifferentiation using time-course chromatin remodeling (ATACseq) and nascent transcription data (PROseq). We stimulated VSMCs from three healthy heart transplant donors with PDGF-BB for 0, 4, 8, 12, and 16 hours and performed sequencing in duplicate for each donor at each time point for a total of 30 PROseq and 30 ATACseq libraries. We identified 231,315 regions of accessible chromatin (peaks) in the ATACseq data and 12,954 genes in the PROseq data across the five time points. Likelihood ratio testing detected 13,285 peaks and 7,438 genes with time-dynamic changes in read counts representing six patterns of variable accessibility and nascent transcription following PDGF-BB stimulation: gradual increase/decrease (I/D), immediate I/D, and transient I/D. Transcription factor binding site (TFBS) motif enrichment of dynamic peaks revealed 31 TFs unique to one time dynamic. We identified 157 genes directly downstream of peaks enriched for the 31 TFBSs sharing similar time-specific fold changes. THAP1 binding site motifs had the highest number of cis-regulatory connections (596) and downstream genes (80). Our results capture the first ever time-course ATACseq and PROseq analysis conducted in human VSMCs and provide the ability to predict regulatory edges between TFs and genes in order to map the regulatory cascading events occurring during VSMC switching from a contractile to a synthetic state.

KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204.

Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target.

Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response.

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/- and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/- aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/- mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/- VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/- aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/- aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/- mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Therapeutic Potential of Targeting p27kip1 in Plaque Vulnerability.

Atherosclerosis, a critical contributor to coronary artery diseases, involves the accumulation of cholesterol, fibrin, and lipids within arterial walls, inciting inflammatory reactions culminating in plaque formation. This multifaceted interplay encompasses excessive fibrosis, fatty plaque development, vascular smooth muscle cell (VSMC) proliferation, and leukocyte migration in response to inflammatory pathways. While stable plaques demonstrate resilience against complications, vulnerable ones, with lipid-rich cores, necrosis, and thin fibrous caps, lead to thrombosis, myocardial infarction, stroke, and acute cerebrovascular accidents. The nuanced phenotypes of VSMCs, modulated by gene regulation and environmental cues, remain pivotal. Essential markers like alpha-SMA, myosin heavy chain, and calponin regulate VSMC migration and contraction, exhibiting diminished expression during VSMC de-differentiation and proliferation. p27kip, a CDK inhibitor, shows promise in regulating VSMC proliferation and appears associated with TNF-α-induced pathways impacting unstable plaques. Oncostatin M (OSM), an IL-6 family cytokine, correlates with MMP upregulation and foam cell formation, influencing plaque development. Efforts targeting mammalian target of rapamycin (mTOR) inhibition, notably using rapamycin and its analogs, demonstrate potential but pose challenges due to associated adverse effects. Exploration of the impact of p27kip impact on plaque macrophages presents promising avenues, yet its complete therapeutic potential remains untapped. Similarly, while OSM has exhibited potential in inducing cell cycle arrest via p27kip, direct links necessitate further investigation. This critical review discusses the role of mTOR, p27kip, and OSM in VSMC proliferation and differentiation followed by the therapeutic potential of targeting these mediators in atherosclerosis to attenuate plaque vulnerability.

Ursolic acid inhibits NLRP3 inflammasome activation and alleviates vascular smooth muscle injury in Kawasaki disease

Kawasaki disease (KD) is a kind of autoimmune disease with systemic vasculitis as the main pathological change. It is critical to explore potential new therapeutic agents to address KD disease and its complications. Ursolic acid (UA) is a pentacyclic triterpene (PT) carboxylic acid that has a number of important pharmacological activities, however, the possible effects of UA on the progression of KD and the mechanism are still unclear. Here we investigated the effects of UA on KD. We revealed that UA improved arterial injury in KD mice. UA improved vascular inflammation in KD mice. In addition, Ursolic suppressed NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation. We further found UA restrained vascular smooth muscle cell (VSMC) dedifferentiation, therefore suppressing KD progression. In summary, UA suppressed NLRP3 inflammasome activation as well as alleviated vascular smooth muscle injury in KD. We thought UA could act as a drug of KD.

HRD1 reduction promotes cholesterol-induced vascular smooth muscle cell phenotypic change via endoplasmic reticulum stress.

Phenotypic change of vascular smooth muscle cells (VSMCs) is the main contributor of vascular pathological remodeling in atherosclerosis. The endoplasmic reticulum (ER) is critical for maintaining VSMC function through elimination of misfolded proteins that impair VSMC cellular function. ER-associated degradation (ERAD) is an ER-mediated process that controls protein quality by clearing misfolded proteins. One of the critical regulators of ERAD is HRD1, which also plays a vital role in lipid metabolism. However, the function of HRD1 in VSMCs of atherosclerotic vessels remains poorly understood. The level of HRD1 expression was analyzed in aortic tissues of mice fed with a high-fat diet (HFD). The H&E and EVG (VERHOEFF'S VAN GIESON) staining were used to demonstrate pathological vascular changes. IF (immunofluorescence) and WB (western blot) were used to explore the signaling pathways in vivo and in vitro. The wound closure and transwell assays were also used to test the migration rate of VSMCs. CRISPR gene editing and transcriptomic analysis were applied in vitro to explore the cellular mechanism. Our data showed significant reduction of HRD1 in aortic tissues of mice under HFD feeding. VSMC phenotypic change and HRD1 downregulation were detected by cholesterol supplement. Transcriptomic and further analysis of HRD1-KO VSMCs showed that HRD1 deficiency induced the expression of genes related to ER stress response, proliferation and migration, but reduced the contractile-related genes in VSMCs. HRD1 deficiency also exacerbated the proliferation, migration and ROS production of VSMCs induced by cholesterol, which promoted the VSMC dedifferentiation. Our results showed that HRD1 played an essential role in the contractile homeostasis of VSMCs by negatively regulating ER stress response. Thus, HRD1 in VSMCs could serve as a potential therapeutic target in metabolic disorder-induced vascular remodeling.

MiR-122 knockdown regulates vascular smooth muscle cells phenotypic switching through enhanced FOXO3 expression.

Vascular smooth muscle cells (VSMCs) phenotypic switching is identified as enhanced dedifferentiation, proliferation, and migration ability of VSMCs, in which microRNAs have been identified as important regulators of the process. The present study is aimed to explore the pathophysiological effect of miR-122 on VSMC phenotypic modulation. Here, the result showed that the decreased miR-122 expression was found in VSMCs subjected to platelet-derived growth factor-BB (PDGF-BB) treatment. Next, we investigated the response of miR-122 knockdown in VSMCs with PDGF-BB stimulation. MiR-122 silencing showed increased proliferation and migration capability, whereas attenuated the differentiation markers expression. The above results were reversed by miR-122 overexpression. Finally, we further demonstrated that FOXO3 was an important target for miR-122. Collectively, we demonstrated that miR-122 silencing promoted VSMC phenotypic modulation partially through upregulated FOXO3 expression that indicated miR-122 may be a novel therapeutic target for neointimal formation.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation

Background and aimsThe pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. MethodsFor in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. ResultsPDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αβ (PDGFRαβ) rather than PDGFRαα or PDGFRββ in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. ConclusionsThe results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

Abstract 17672: Implications of Pcsk9 on Abdominal Aortic Aneurysm

Objective: Recent genome-wide association studies revealed a potential role of proprotein convertase subtilisin/kexin type9(Pcsk9) in the human abdominal aortic aneurysm(AAA) disease.Altered Pcsk9 gene expression and smaller AAA diameter were observed in murine AAA using the porcine pancreatic elastase(PPE) model in our lab. Pcsk9 increases plasma LDL-cholesterol,enhances tissue inflammation and induces vascular smooth muscle cell (VSMC) senescence, hallmarks in AAA.However, the exact cellular mechanisms of Pcsk9 in AAA disease remain unclear.Despite the absence of a pharmacological treatment for AAA, monoclonal Pcsk9 antibody therapies have proven effective against statin-resistant hypercholesterolemia.This study aimed to explore the impacts of Pcsk9 on aortic vascular inflammation and VSMC dedifferentiation with a view to better understanding AAA pathophysiology and providing a basis for future investigations into Pcsk9 inhibitors. Hypothesis: We hypothesized that local vascular injury escalates Pcsk9 expression,amplifying vascular inflammation and driving VSMC towards dedifferentiation that further advances AAA. Conversely,Pcsk9 inhibition will mitigate these processes. Methods: AAA was induced by PPE model in wildtype C57Bl/6 mice and Pcsk9 knock-out (KO)mice.Sonography was used to evaluate the aneurysm growth. Single-cell RNA sequencing was employed to examine local changes in the aortic wall. Local vascular Pcsk9,pro-inflammatory and VSMC marker gene/protein expression were quantified using qPCR and immunohistochemistry.VSMCs were treated with exogenous Pcsk9 in vitro to assess metabolism via Seahorse. Results: Reduced AAA diameters were observed in Pcsk9 KO mice compared to wildtype controls. Significant upregulation of local Pcsk9 expression accompanied AAA induction. Single-cell RNA sequencing revealed altered gene expression profiles across vascular cell populations. In vitro,VSMCs displayed changes in marker gene/protein expression after treatment with exogenous Pcsk9, alongside metabolic changes (Mann-Whitney Test). Conclusions: The findings suggest a significant role of Pcsk9 in AAA pathogenesis. Its inhibition appears to mitigate AAA growth,indicating a promising treatment target for AAA.

Myeloid-derived growth factor suppresses VSMC dedifferentiation and attenuates postinjury neointimal formation in rats by activating S1PR2 and its downstream signaling.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 μg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature.

Hsa_circ_0031891 targets miR-579-3p to enhance HMGB1 expression and regulate PDGF-BB-induced human aortic vascular smooth muscle cell proliferation, migration, and dedifferentiation.

Atherosclerosis (AS) is an underlying cause of the majority of coronary artery disease (CAD), in which proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) exert vital roles. It has been reported that circular RNAs (circRNAs) are associated with the VSMCs function. Here, we undertook to explore the biological function and mechanism of hsa_circ_0031891 in a platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model. Hsa_circ_0031891 and microRNA-579-3p (miR-579-3p) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation and migration were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assay. Protein levels of alpha-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), Osteopontin, and High mobility group box-1 (HMGB1) were determined using western blot assay. After predicting via a variety of bioinformatics software, the binding between miR-579-3p and hsa_circ_0031891 or HMGB1 was validated using dual-luciferase reporter and RNA pull-down assays. Increased hsa_circ_0031891 and HMGB1 and reduced miR-579-3p were found in CAD patients and PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs). Moreover, hsa_circ_0031891 deficiency relieved PDGF-BB-mediated HA-VSMC proliferation, migration, and dedifferentiation. Mechanically, hsa_circ_0031891 modulated HMGB1 expression via sponging miR-579-3p. Hsa_circ_0031891 boosted PDGF-BB-induced proliferation, migration, and dedifferentiation partly by regulating the miR-579-3p/HMGB1 axis, hinting at a feasible therapeutic strategy for AS.