Smooth Muscle Cell Phenotypic Modulation Research Articles

Stress, Vascular Smooth Muscle Cell Phenotype and Atherosclerosis: Novel Insight into Smooth Muscle Cell Phenotypic Transition in Atherosclerosis.

Our work is to establish more distinct association between specific stress and vascular smooth muscle cells (VSMCs) phenotypes to alleviate atherosclerotic plaque burden and delay atherosclerosis (AS) progression. In recent years, VSMCs phenotypic transition has received significant interests. Different stresses were found to be associated with VSMCs phenotypic transition. However, the explicit correlation between VSMCs phenotype and specific stress has not been elucidated clearly yet. We discover that VSMCs phenotypic transition, which is widely involved in the progression of AS, is associated with specific stress. We discuss approaches targeting stresses to intervene VSMCs phenotypic transition, which may contribute to develop innovative therapies for AS.

Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm

BackgroundThe fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood.MethodsIn this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRβ, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib.ResultsSingle-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation.ConclusionOur findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

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 18578: BMP9 Regulates Endothelial Expression of Mediators of Pulmonary Vascular Remodeling

Background: Loss-of-function mutations in GDF2 /BMP9 are found in heritable pulmonary arterial hypertension (PAH), suggesting a protective role, while protective or pathogenic effects are found in experimental PAH studies depending on context. The net contribution of BMP9 in PAH thus remains controversial. Hypothesis: BMP9 regulates the secretome of pulmonary microvascular endothelium (PMVEC) to contribute to pulmonary artery smooth muscle cell (PASMC) phenotype modulation. Methods: We tested recombinant BMP9, BMP9/BMP10 trap ALK1-Fc, and BMP9 neutralizing antibody before and after development of pulmonary hypertension (PH) in distinct experimental models. We analyzed the impact of BMP9 on the PMVEC transcriptome, intersection with human and experimental PAH lung transcriptomes, and tested the impact of EC-derived genes on PASMC phenotype and growth. Results: ALK1-Fc and anti-BMP9 prevented or reversed experimental PH administered before or after onset of experimental PH. Recombinant disulfide-linked BMP9 was not protective in SUGEN/Hypoxia (SU-Hx)- or monocrotaline (MCT)-induced experimental PH, in contrast to previous studies using incompletely disulfide-linked BMP9, suggesting dissociated monomers might function as competitive BMP9 antagonists. Consistent with this interpretation, BMP9 upregulated PMVEC genes associated with pulmonary vascular remodeling in experimental and human PAH including CXCL12, IGFBP4, ET-1, VEGF-A, and PDGF-BB. Stimulation of PMVEC with BMP9 induced a contractile phenotype ( CNN1;TAGLN ) in co-cultured PASMC. Administration of ALK1-Fc or anti-BMP9 attenuated expression of several of these growth factors in SU-Hx PH. In experimental PH ALK1-Fc and ACTRIIA-Fc elicited peripheral edema, a phenotype associated with use of ALK1-Fc in human trials. Conclusions: BMP9 contributes to experimental PH by regulating expression of EC-derived molecules modulating PASMC growth and phenotype. In contrast to broad strategies that inhibit BMP9 and BMP10 associated with HHT mimicry in clinical trials and rodent PH models, treatment with anti-BMP9 was well-tolerated. Selective antagonism of BMP9 represents a potential therapeutic strategy for human PAH.

Abstract 12901: ADAR1 RNA Editing in Vascular Smooth Muscle Maintains Vascular Integrity, Regulates Cell State Transition, and is a Novel Mechanism of Coronary Artery Disease Risk

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) molecules to prevent the dsRNA sensor, MDA5 ( IFIH1 ), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). Here, we reveal in human atherosclerosis, smooth muscle cells (SMCs) express immunogenic dsRNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). Loss of RNA editing through ADAR1 KD in HCASMC causes an ISG response characterized by upregulation of ISG genes (i.e. ISG15, IFI6, IFIH1 ) in addition to genes with known roles in SMC phenotypic transition and CAD risk (i.e. KLF4 , TCF21 , and ZEB2 ). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro and in atherosclerosis prone mice. scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype. To further evaluate the role for SMC RNA editing in vivo , we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ). At 2 weeks following SMC- Adar1 KD, we observe 50% mortality, where mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model ( Adar1 flox/flox , Cx40 CreERT2 ). scRNAseq of aortic tissue shows that SMC- Adar1 KD causes ISG activation throughout the vessel wall, SMCs show loss of mature SMC markers, and there is robust macrophage and T cell infiltration. Through evaluation of ligand-receptor interaction, we show that Ccl5:Ccr5 is the principal regulator of cell infiltration following SMC- Adar1 KD. Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.

Causal Role for Neutrophil Elastase in Thoracic Aortic Dissection in Mice.

Thoracic aortic dissection (TAD) is a life-threatening aortic disease without effective medical treatment. Increasing evidence has suggested a role for NE (neutrophil elastase) in vascular diseases. In this study, we aimed at investigating a causal role for NE in TAD and exploring the molecular mechanisms involved. β-aminopropionitrile monofumarate was administrated in mice to induce TAD. NE deficiency mice, pharmacological inhibitor GW311616A, and adeno-associated virus-2-mediated in vivo gene transfer were applied to explore a causal role for NE and associated target gene in TAD formation. Multiple functional assays and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in TAD. NE aortic gene expression and plasma activity was significantly increased during β-aminopropionitrile monofumarate-induced TAD and in patients with acute TAD. NE deficiency prevents β-aminopropionitrile monofumarate-induced TAD onset/development, and GW311616A administration ameliorated TAD formation/progression. Decreased levels of neutrophil extracellular traps, inflammatory cells, and MMP (matrix metalloproteinase)-2/9 were observed in NE-deficient mice. TBL1x (F-box-like/WD repeat-containing protein TBL1x) has been identified as a novel substrate and functional downstream target of NE in TAD. Loss-of-function studies revealed that NE mediated inflammatory cell transendothelial migration by modulating TBL1x-LTA4H (leukotriene A4 hydrolase) signaling and that NE regulated smooth muscle cell phenotype modulation under TAD pathological condition by regulating TBL1x-MECP2 (methyl CpG-binding protein 2) signal axis. Further mechanistic studies showed that TBL1x inhibition decreased the binding of TBL1x and HDAC3 (histone deacetylase 3) to MECP2 and LTA4H gene promoters, respectively. Finally, adeno-associated virus-2-mediated Tbl1x gene knockdown in aortic smooth muscle cells confirmed a regulatory role for TBL1x in NE-mediated TAD formation. We unravel a critical role of NE and its target TBL1x in regulating inflammatory cell migration and smooth muscle cell phenotype modulation in the context of TAD. Our findings suggest that the NE-TBL1x signal axis represents a valuable therapeutic for treating high-risk TAD patients.

N-acetylcysteine ameliorates erectile dysfunction in rats with hyperlipidemia by inhibiting oxidative stress and corpus cavernosum smooth muscle cells phenotypic modulation.

Hyperlipidemia is a major risk factor for erectile dysfunction (ED). Oxidative stress and phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMCs) are the key pathological factors of ED. N-acetylcysteine (NAC) can inhibit oxidative stress; however, whether NAC can alleviate pathological variations in the corpus cavernosum and promote erectile function recovery in hyperlipidemic rats remains unclear. A hyperlipidemia model was established using 27 eight-week-old male Sprague-Dawley (SD) rats fed a high-fat and high-cholesterol diet (hyperlipidemic rats, HR). In addition, 9 male SD rats were fed a normal diet to serve as controls (NC). HR rats were divided into three groups: HR, HR+normal saline (NS), and HR+NAC (n = 9 for each group; NS or NAC intraperitoneal injections were administered daily for 16 weeks). Subsequently, the lipid profiles, erectile function, oxidative stress, phenotypic modulation markers of CCSMCs, and tissue histology were analyzed. The experimental results revealed that erectile function was significantly impaired in the HR and HR + NS groups, but enhanced in the HR + NAC group. Abnormal lipid levels, over-activated oxidative stress, and multi-organ lesions observed in the HR and HR + NS groups were improved in the HR + NAC group. Moreover, the HR group showed significant phenotypic modulation of CCSMCs, which was also inhibited by NAC treatment. This report focuses on the therapeutic effect of NAC in restoring erectile function using a hyperlipidemic rat model by preventing CCSMC phenotypic modulation and attenuating oxidative stress.

Early clinical outcomes and molecular smooth muscle cell phenotyping using a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome

ObjectivesPatients with Loeys-Dietz syndrome demonstrate a heightened risk of distal thoracic aortic events after valve-sparing aortic root replacement. This study assesses the clinical risks and hemodynamic consequences of a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome and characterizes smooth muscle cell phenotype in Loeys-Dietz syndrome aneurysmal and normal-sized downstream aorta. MethodsPatients with genetically confirmed Loeys-Dietz syndrome (n = 8) underwent prophylactic aortic arch replacement during valve-sparing aortic root replacement. Four-dimensional flow magnetic resonance imaging studies were performed in 4 patients with Loeys-Dietz syndrome (valve-sparing aortic root replacement + arch) and compared with patients with contemporary Marfan syndrome (valve-sparing aortic root replacement only, n = 5) and control patients (without aortopathy, n = 5). Aortic tissues from 4 patients with Loeys-Dietz syndrome and 2 organ donors were processed for anatomically segmented single-cell RNA sequencing and histologic assessment. ResultsPatients with Loeys-Dietz syndrome valve-sparing aortic root replacement + arch had no deaths, major morbidity, or aortic events in a median of 2 years follow-up. Four-dimensional magnetic resonance imaging demonstrated altered flow parameters in patients with postoperative aortopathy relative to controls, but no clear deleterious changes due to arch replacement. Integrated analysis of aortic single-cell RNA sequencing data (>49,000 cells) identified a continuum of abnormal smooth muscle cell phenotypic modulation in Loeys-Dietz syndrome defined by reduced contractility and enriched extracellular matrix synthesis, adhesion receptors, and transforming growth factor-beta signaling. These modulated smooth muscle cells populated the Loeys-Dietz syndrome tunica media with gradually reduced density from the overtly aneurysmal root to the nondilated arch. ConclusionsPatients with Loeys-Dietz syndrome demonstrated excellent surgical outcomes without overt downstream flow or shear stress disturbances after concomitant valve-sparing aortic root replacement + arch operations. Abnormal smooth muscle cell–mediated aortic remodeling occurs within the normal diameter, clinically at-risk Loeys-Dietz syndrome arch segment. These initial clinical and pathophysiologic findings support concomitant arch replacement in Loeys-Dietz syndrome.

The Maastricht Acquisition Platform for Studying Mechanisms of Cell-Matrix Crosstalk (MAPEX): An Interdisciplinary and Systems Approach towards Understanding Thoracic Aortic Disease.

Current management guidelines for ascending thoracic aortic aneurysms (aTAA) recommend intervention once ascending or sinus diameter reaches 5-5.5 cm or shows a growth rate of >0.5 cm/year estimated from echo/CT/MRI. However, many aTAA dissections (aTAAD) occur in vessels with diameters below the surgical intervention threshold of <55 mm. Moreover, during aTAA repair surgeons observe and experience considerable variations in tissue strength, thickness, and stiffness that appear not fully explained by patient risk factors. To improve the understanding of aTAA pathophysiology, we established a multi-disciplinary research infrastructure: The Maastricht acquisition platform for studying mechanisms of tissue-cell crosstalk (MAPEX). The explicit scientific focus of the platform is on the dynamic interactions between vascular smooth muscle cells and extracellular matrix (i.e., cell-matrix crosstalk), which play an essential role in aortic wall mechanical homeostasis. Accordingly, we consider pathophysiological influences of wall shear stress, wall stress, and smooth muscle cell phenotypic diversity and modulation. Co-registrations of hemodynamics and deep phenotyping at the histological and cell biology level are key innovations of our platform and are critical for understanding aneurysm formation and dissection at a fundamental level. The MAPEX platform enables the interpretation of the data in a well-defined clinical context and therefore has real potential for narrowing existing knowledge gaps. A better understanding of aortic mechanical homeostasis and its derangement may ultimately improve diagnostic and prognostic possibilities to identify and treat symptomatic and asymptomatic patients with existing and developing aneurysms.

Structural Fragility of Cerebral Aneurysms: A Comparative Analysis of Unruptured &amp; Ruptured Aneurysms

Ongoing studies to understand the mechanisms behind intracranial aneurysm (IA) pathogenesis characterize intracranial aneurysm as a maladaptive chronic inflammatory disease. An overwhelmingly large number of studies implicate inflammation as a crucial driver of aneurysmal growth and attendant rupture. The induction of aneurysmal formation begins with endothelial damage, which secondarily activates a progressive inflammatory response that compromises the structural integrity of the vessel wall. Pivotal consequences of this inflammatory response is a disintegration of the internal elastic lamina, myointimal hyperplasia, and vascular smooth muscle cell (vsmc) phenotypic modulation and subsequent apoptosis. Interestingly, analysis of gene expression profiles in patients with intracranial aneurysms commonly highlight a differential expression of genes related to inflammatory/immune responses, cell adhesion, and muscle contraction. A detailed understanding of intracranial aneurysmal pathogenesis may be possible by analyzing morphological differences in aneurysmal structure between unruptured and ruptured lesions. As such, in this review, we have attempted to understand sequential adaptations of the aneurysmal wall during various periods of growth, development and rupture by providing evidence for structural, genetic, and molecular differences between unruptured and ruptured intracranial aneurysm.

Smooth muscle α-actin missense variant promotes atherosclerosis through modulation of intracellular cholesterol in smooth muscle cells.

The variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis. Apoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice. These data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden.

MiR-342-5p promotes vascular smooth muscle cell phenotypic transition through a negative-feedback regulation of Notch signaling via targeting FOXO3

AimUnder various pathological conditions such as cancer, vascular smooth muscle cells (vSMCs) transit their contractile phenotype into phenotype(s) characterized by proliferation and secretion, a process called vSMC phenotypic transition (vSMC-PT). Notch signaling regulates vSMC development and vSMC-PT. This study aims to elucidate how the Notch signal is regulated. Main methodsGene-modified mice with a SM22α-CreERT2 transgene were generated to activate/block Notch signaling in vSMCs. Primary vSMCs and MOVAS cells were cultured in vitro. RNA-seq, qRT-PCR and Western blotting were used to evaluated gene expression level. EdU incorporation, Transwell and collagen gel contraction assays were conducted to determine the proliferation, migration and contraction, respectively. Key findingsNotch activation upregulated, while Notch blockade downregulated, miR-342-5p and its host gene Evl in vSMCs. However, miR-342-5p overexpression promoted vSMC-PT as shown by altered gene expression profile, increased migration and proliferation, and decreased contraction, while miR-342-5p blockade exhibited the opposite effects. Moreover, miR-342-5p overexpression significantly suppressed Notch signaling, and Notch activation partially abolished miR-342-5p-induced vSMC-PT. Mechanically, miR-342-5p directly targeted FOXO3, and FOXO3 overexpression rescued miR-342-5p-induced Notch repression and vSMC-PT. In a simulated tumor microenvironment, miR-342-5p was upregulated by tumor cell-derived conditional medium (TCM), and miR-342-5p blockade abrogated TCM-induced vSMC-PT. Meanwhile, conditional medium from miR-342-5p-overexpressing vSMCs significantly enhanced tumor cell proliferation, while miR-342-5p blockade had the opposite effects. Consistently, in a co-inoculation tumor model, miR-342-5p blockade in vSMCs significantly delayed tumor growth. SignificancemiR-342-5p promotes vSMC-PT through a negative-feedback regulation of Notch signaling via downregulating FOXO3, which could be a potential target for cancer therapy.

Abstract 703: Dioxin-induced Smooth Muscle Cell Phenotypic Modulation And Atherosclerosis Worsens In The Absence Of AHR

Background: Dioxin (TCDD, 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin) is an active constituent of tobacco smoke and air pollution that activates the aryl hydrocarbon receptor (AHR). Dioxin has been linked to an increased risk of coronary artery disease (CAD). However, the corresponded molecular mechanisms remain unclear. Smooth muscle cells (SMC) play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. In this study, we aimed to understand the role of AHR in modulating the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: SMC lineage-tracing (SMC Lin ) mice ( Tg Myh11-CreERT2 , ROSA tdT/tdT , ApoE -/- ) without any TCDD injection were used as control. TCDD were injected to both SMC Lin mice, and SMC-specific Ahr knockout mice. High fat diet (HFD) and TCDD injection started at 8 weeks of age and aortic roots were harvested at 24 weeks of age for either histology or single-cell sequencing (10X Genomics, triplicates). Histology analysis was performed at the aortic sinus of control mice (n=9), TCDD injected SMC Lin mice (n=12) and TCDD injected SMC-specific Ahr knockout mice (n=9). Immunohistochemistry was performed with anti-SM22α antibody and CD68 antibody. Results: The histological analysis of the aortic sinus showed increased lesion area after TCDD treatment in the SMC Lin control mice. In the SMC-specific knockout of Ahr , the lesion area was significantly larger compared to the SMC Lin control mice when treated with TCDD along with significantly increased presence of Tagln + cells in the lesion. These results indicated TCDD leads to adverse remodeling of atherosclerosis and that the presence of Ahr gene is protective against the athero-promoting effects of dioxin. The scRNA-seq of SMC Lin control mice treated with TCDD exposure showed that the SMC phenotypic modulation was markedly inhibited, leading to the presence of fewer fibromyocytes (FMC). Increased FMC to chondromyocytes (CMC) modulation resulted in further reduction of FMC population in the Ahr -KO mice treated with TCDD. Conclusion: Dioxin adversely remodels atherosclerotic plaque, and the lack of AHR in SMC can exacerbate the atherosclerotic process, suggesting that dioxin may exhibit its harmful effect via non-AHR dependent pathway.

Abstract 341: Study Of ADAR1 Implicates RNA Editing In Vascular Smooth Muscle Cells As A Mechanism Of Coronary Artery Disease Risk

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit double stranded RNA (dsRNA) molecules to prevent activation of the dsRNA sensor MDA5 ( IFIH1 ) that stimulates an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). We hypothesize that smooth muscle cell (SMC) specific RNA editing is required to prevent vascular calcification and inflammation. Here, we demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). ADAR1 KD in HCASMC causes an ISG response characterized by massive upregulation of key ISG genes (e.g. ISG15, IFI6, IFIH1 ). Importantly, ADAR1 KD stimulates the upregulation of genes with known roles in SMC phenotypic transition and CAD risk, including KLF4 , TCF21 , ATF3 , and ZEB2 . This effect is dependent on activation of MDA5. Additionally, we demonstrate ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro where ADAR1 KD inhibits SMC maturation and dysregulates the calcification gene program in an MDA5 dependent manner. Using scRNAseq data collected from human vascular tissue we show that immunogenic dsRNA are disproportionately expressed in human SMC in atherosclerosis. Similarly, in atherosclerosis prone mice, scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype — consistent with MDA5 activation. To further evaluate the role for SMC specific RNA editing in vivo, we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ ). At 2 weeks following SMC- Adar1 KD, mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model ( Adar1 flox/flox , Cx40 CreERT2 ). Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.

Abstract 284: Pro-Inflammatory Adventitial Fibroblast Contribute To Aortic Aneurysms In Loeys Dietz Syndrome

Introduction: Vascular smooth muscle cell (SMC) phenotype modulation is implicated in thoracic aneurysm formation, however, the role of adventitial fibroblasts (AdvFib) in aneurysm formation remains poorly characterized. Loeys-Dietz Syndrome (LDS), caused by a spectrum of mutations in the TGF-β signaling cascade, leads to aggressive thoracic aneurysms. Given the ubiquitous nature of TGF-β signaling machinery, the exact cell type(s) responsible for initiation of LDS pathogenesis is poorly understood. Methods: We performed single cell transcriptomic characterization of aortic root and ascending aortic samples from LDS patients ((n=6 LDS (harboring TGFB2 , TGFBR1 , TGFBR2 , or SMAD3 mutation); n=2 Donor control)) as well as root/ascending aorta from a murine model of LDS ( Tgfbr2 G357W/+ ) to understand cell-state transitions and transcriptomic alterations in LDS. Results: Single cell RNA sequencing (scRNAseq) of human and mice LDS samples (in total &gt;40,000 cells) revealed a secondary pattern of cellular activation in LDS as compared to Marfan Syndrome. Instead of SMC phenotypic modulation as seen in MFS, LDS appears to preferentially modulate adventitial fibroblasts (AdvFib) phenotype in the Tgfbr2 G357W/+ mouse model. Human LDS patient’s AdvFib turn on high levels of macrophage-recruiting pro-inflammatory cytokines CCL2 and HLA-B, with a corresponding 1.8-fold increase in LDS aortic wall macrophages compared to Donor. A more extreme pattern was observed in the Tgfbr2 G357W/+ LDS mouse model. While Tgfbr2 G357W/+ SMC are transcriptomically similar to wild type counterparts, AdvFib were activated into a pro-inflammatory state associated with increased aortic macrophage recruitment ( Ccl2 , Il6 , Vcam1, and Cxcl2 ) and fibrotic response ( Col1a1 , Col1a2 , Col3a1, and Fn1 ). A 6-fold increase in the number of aortic wall macrophages was identified in Tgfbr2 G357W/+ compared to WT. Conclusions: LDS mouse and human AdvFib undergo phenotypic alterations into a pro-inflammatory state evidenced by increased expression of numerous cytokines, macrophage recruitment, and ECM deposition. AdvFib, in addition to SMCs, are potentially another important pathological cell population in LDS aneurysm formation.

Smooth Muscle Cell Klf4 Expression Is Not Required for Phenotype Modulation or Aneurysm Formation in Marfan Syndrome Mice-Brief Report.

Smooth muscle cell (SMC) phenotypic reprogramming toward a mixed synthetic-proteolytic state is a central feature of aortic root aneurysm in Marfan syndrome (MFS). Previous work identified Klf4 as a potential mediator of SMC plasticity in MFS. MFS (Fbn1C1041G/+) mouse strains with an inducible vascular SMC fluorescent reporter (MFSSMC) with or without SMC-specific deletion of Klf4 exons 2 to 3 (MFSSMC-Klf4Δ) were generated. Simultaneous SMC tracing and Klf4 loss-of-function (Klf4Δ mice) was induced at 6 weeks of age. Aneurysm growth was assessed via serial echocardiography (4-24 weeks). Twenty-four-week-old mice were assessed via histology, RNA in situ hybridization, and aortic single-cell RNA sequencing. MFS mice demonstrated progressive aortic root dilatation compared with control (WTSMC) mice regardless of Klf4 genotype (P<0.001), but there was no difference in aneurysm growth in MFSSMC-Klf4Δ versus MFSSMC (P=0.884). Efficient SMC Klf4 deletion was confirmed via lineage-stratified genotyping, RNA in situ hybridization, and immunohistochemistry. Single-cell RNA sequencing of traced SMCs revealed a highly similar pattern of phenotype modulation marked by loss of contractile markers (eg, Myh11, Cnn1) and heightened expression of matrix genes (eg, Col1a1, Fn1) between Klf4 genotypes. Pseudotemporal quantitation of SMC dedifferentiation confirmed that Klf4 deletion did not alter the global extent of phenotype modulation, but reduced expression of 23 genes during this phenotype transition in MFSSMC-Klf4Δmice, including multiple chondrogenic genes expressed by only the most severely dedifferentiated SMCs (eg, Cytl1, Tnfrsf11b). Klf4 is not required to initiate SMC phenotype modulation in MFS aneurysm but may exert regulatory control over chondrogenic genes expressed in highly dedifferentiated SMCs.

Meet the First Authors.

Meet the First Authors.

Age-dependent phenotypic modulation of smooth muscle cells in the normal ascending aorta.

Ascending aortic aneurysms are associated with pre-existing conditions, including connective tissue disorders (i.e., Marfan syndrome) and bicuspid aortic valves. The underlying mechanisms remain uncertain. Even less is known regarding ascending aortic aneurysms in individuals with normal (i.e., tricuspid) aortic valves (TAV), and without known aneurysm-associated disorders. Regardless of etiology, the risk of aortic complications increases with biological age. Phenotypic modulation of smooth muscle cells (SMCs) is a feature of ascending aortic aneurysms, whereby contractile SMCs are replaced with synthetic SMCs that are capable of degrading the aortic wall. We asked whether age itself causes dysfunctional SMC phenotype modulation, independent of aortic dilatation or pre-existing aneurysm-associated diseases. Non-dilated ascending aortic samples were obtained intra-operatively from 40 patients undergoing aortic valve surgery (range: 20-82 years old, mean: 59.1 ± 15.2). Patients with known genetic diseases or aortic valve malformations were excluded. Tissue was divided, and a portion was formalin-fixed and immunolabeled for alpha-smooth muscle actin (ASMA), a contractile SMC protein, and markers of synthetic (vimentin) or senescent (p16/p21) SMCs. Another fragment was used for SMC isolation (n = 10). Cultured SMCs were fixed at cell passage 2 and stained for phenotype markers, or were cultured indefinitely to determine replicative capacity. In whole tissue, ASMA decreased (R2 = 0.47, P < 0.0001), while vimentin increased (R2 = 0.33, P = 0.02) with age. In cultured SMCs, ASMA decreased (R2 = 0.35, P = 0.03) and vimentin increased (R2 = 0.25, P = 0.04) with age. p16 (R2 = 0.34, P = 0.02) and p21 (R2 = 0.29, P = 0.007) also increased with age in SMCs. Furthermore, the replicative capacity of SMCs from older patients was decreased compared to that of younger patients (P = 0.03). By investigating non-dilated aortic samples from individuals with normal TAVs, we found that age itself has a negative impact on SMCs in the ascending aortic wall, whereby SMCs switched from the contractile phenotype to maladaptive synthetic or senescent states with increased age. Therefore, based on our findings, modification of SMC phenotype should be studied as a therapeutic consideration against aneurysms in the future, regardless of etiology.

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.