Vascular Smooth Muscle Cells Markers Research Articles

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.

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.

Low concentrations of TNF-α invitro transform the phenotype of vascular smooth muscle cells and enhance their survival in a three-dimensional culture system.

Vascular smooth muscle cells (VSMCs) are commonly used as seed cells in tissue-engineered vascular constructions. However, their variable phenotypes and difficult to control functions pose challenges. This study aimed to overcome these obstacles using a three-dimensional culture system. Calf VSMCs were administered tumor necrosis factor-alpha (TNF-α) before culturing in two- and three-dimensional well plates and polyglycolic acid (PGA) scaffolds, respectively. The phenotypic markers of VSMCs were detected by immunofluorescence staining and western blotting, and the proliferation and migration abilities of VSMCs were detected by CCK-8, EDU, cell counting, scratch, and Transwell assays. TNF-α rapidly decreased the contractile phenotypic markers and elevated the synthetic phenotypic markers of VSMCs, as well as markedly increasing the proliferation and migration ability of VSMCs under two- and three-dimensional culture conditions. TNF-α can rapidly induce a phenotypic shift in VSMCs and change their viability on PGA scaffolds.

LncRNA CARMN inhibits abdominal aortic aneurysm formation and vascular smooth muscle cell phenotypic transformation by interacting with SRF

Phenotypic transformation of vascular smooth muscle cells (VSMCs) plays a crucial role in abdominal aortic aneurysm (AAA) formation. CARMN, a highly conserved, VSMC-enriched long noncoding RNA (lncRNA), is integral in orchestrating various vascular pathologies by modulating the phenotypic dynamics of VSMCs. The influence of CARMN on AAA formation, particularly its mechanisms, remains enigmatic. Our research, employing single-cell and bulk RNA sequencing, has uncovered a significant suppression of CARMN in AAA specimens, which correlates strongly with the contractile function of VSMCs. This reduced expression of CARMN was consistent in both 7- and 14-day porcine pancreatic elastase (PPE)-induced mouse models of AAA and in human clinical cases. Functional analyses disclosed that the diminution of CARMN exacerbated PPE-precipitated AAA formation, whereas its augmentation conferred protection against such formation. Mechanistically, we found CARMN's capacity to bind with SRF, thereby amplifying its role in driving the transcription of VSMC marker genes. In addition, our findings indicate an enhancement in CAMRN transcription, facilitated by the binding of NRF2 to its promoter region. Our study indicated that CARMN plays a protective role in preventing AAA formation and restrains the phenotypic transformation of VSMC through its interaction with SRF. Additionally, we observed that the expression of CARMN is augmented by NRF2 binding to its promoter region. These findings suggest the potential of CARMN as a viable therapeutic target in the treatment of AAA.Graphical abstract

Abstract WMP118: Utilizing Patient-Derived Induced Pluripotent Stem Cells as a Cellular Model for Studying Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a rare cerebrovascular disease that can lead to stroke. A histopathological characteristic in MMD is the thickening of the intimal layer that causes narrowing of the vessel, suggesting dysfunctions in the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The mechanism of MMD is largely unknown, due to a lack of established MMD-specific cellular or animal models. In this study, we established patient-derived cellular co-culture models using MMD iPSC-derived ECs and VSMCs. We also generated a 3D vascular organoid model to more closely mimic the MMD vasculature and function. Methods: Peripheral blood mononuclear cells (PBMCs) from MMD patients or healthy controls were used to generate iPSCs (n=4 per group). Differentiated ECs and VSMCs were validated and their functional characteristics were investigated in single and co-cultures. For co-cultures, ECs and VSMCs were labeled with Vibrant cell labeling dyes (DiO and DiD), followed by either transwell migration assay (ECs on bottom of cell culture plate and VSMCs on top Transwell insert) or in vitro angiogenic tube formation assay. Control and MMD iPSCs were used to generate 3D cellular organoids, and their cellular characteristics were examined using immunohistochemistry with antibodies labeling ECs and VSMCs. Results: While single cultures of ECs displayed similar tube formation patterns between control and MMD, co-cultures of ECs with VSMCs showed that MMD ECs failed to stabilize in vitro angiogenic tubes and formed aggregates at 24hr, when compared to control ECs. Transwell migration assays showed MMD VSMCs migrated faster than control VSMCs. Immunostaining of organoids with EC and VSMC markers showed notable differences in cellular composition, with MMD organoid showing compromised integrity of vascular networks, when compared with controls. Conclusions: We have established 3D organoid cellular models and in vitro co-cultures using MMD patient-derived iPSCs. Our co-culture studies revealed functional impairment in MMD EC/VSMC and organoid models revealed distinct characteristics in their vascular networks, possibly contributing to MMD pathology. These results shed useful insight into the cellular and molecular mechanisms driving MMD.

Extracellular Histones Promote Calcium Phosphate-Dependent Calcification in Mouse Vascular Smooth Muscle Cells.

Vascular calcification, a major risk factor for cardiovascular events, is associated with a poor prognosis in chronic kidney disease (CKD) patients. This process is often associated with the transformation of vascular smooth muscle cells (VSMCs) into cells with osteoblast-like characteristics. Damage-associated molecular patterns (DAMPs), such as extracellular histones released from damaged or dying cells, are suspected to accumulate at calcification sites. To investigate the potential involvement of DAMPs in vascular calcification, we assessed the impact of externally added histones (extracellular histones) on calcium and inorganic phosphate-induced calcification in mouse VSMCs. Our study found that extracellular histones intensified calcification. We also observed that the histones decreased the expression of VSMC marker genes, while simultaneously increasing the expression of osteoblast marker genes. Additionally, histones treated with DNase I, which degrades dsDNA, attenuated this calcification, compared with the non-treated histones, suggesting a potential involvement of dsDNA in this process. Elevated levels of dsDNA were also detected in the serum of CKD model mice, underlining its potential role in vascular calcification in CKD. Our findings suggest that extracellular histones could play a pivotal role in the vascular calcification observed in CKD.

Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3-7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

Bone Morphogenetic Protein-4 Promotes Phenotypic Modulation via SMAD-4/MCT-4 Axis in Vascular Smooth Muscle Cells

Introduction: This study aimed to determine whether bone morphogenetic protein-4 (BMP-4), which increases in response to intimal hyperplasia, promotes phenotype transition in vascular smooth muscle cells (VSMCs). Methods: Balloon injury was used to induce intimal hyperplasia in rats. Hematoxylin-eosin staining was used to detect the alteration of vascular structure. Serum levels of BMP-4 and lactate were detected by ELISA. Human aortic smooth muscle cells (HA-SMCs) were cultured. Protein and mRNA expression levels were detected through Western blot and real-time PCR. Cell migration was measured by transwell assay. Results: Our data showed that serum concentration of BMP-4 was upregulated after balloon injury. Treatment with BMP-4 inhibitor DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo(1,5-a)pyrimidin-3-yl)quinoline) suppressed the abnormal expression of BMP-4 and inhibited the intimal hyperplasia induced by balloon injury. Compared to BMP-4-negative medium, BMP-4-positive medium was associated with higher synthetic VSMC marker expression levels and lower in contractile gene markers in cultured HA-SMCs. Transfection of monocarboxylic acid transporters-4 (MCT-4) siRNA inhibited the excretion of lactate induced by BMP-4. Conclusion: Our analyses provided evidence that BMP-4 and its regulator Smad-4 are key regulators in MCT-4-mediated lactate excretion. This indicates that BMP-4 stimulates the phenotypic transition of VSMCs via SMAD-4/MCT-4 signaling pathway.

PURPL and NEAT1 Long Non-Coding RNAs Are Modulated in Vascular Smooth Muscle Cell Replicative Senescence.

Cellular senescence is characterized by proliferation and migration exhaustion, senescence-associated secretory phenotype (SASP), and oxidative stress. Senescent vascular smooth muscle cells (VSMCs) contribute to cardiovascular diseases and atherosclerotic plaque instability. Since there are no unanimously agreed senescence markers in human VSMCs, to improve our knowledge, we looked for new possible senescence markers. To this end, we first established and characterized a model of replicative senescence (RS) in human aortic VSMCs. Old cells displayed several established senescence-associated markers. They stained positive for the senescence-associated β-galactosidase, showed a deranged proliferation rate, a dramatically reduced expression of PCNA, an altered migratory activity, increased levels of TP53 and cell-cycle inhibitors p21/p16, and accumulated in the G1 phase. Old cells showed an altered cellular and nuclear morphology, downregulation of the expression of LMNB1 and HMGB1, and increased expression of SASP molecules (IL1β, IL6, IL8, and MMP3). In these senescent VSMCs, among a set of 12 manually selected long non-coding RNAs (lncRNAs), we detected significant upregulation of PURPL and NEAT1. We observed also, for the first time, increased levels of RRAD mRNA. The detection of modulated levels of RRAD, PURPL, and NEAT1 during VSMC senescence could be helpful for future studies on potential anti-aging factors.

BACH1 regulates the differentiation of vascular smooth muscle cells from human embryonic stem cells via CARM1-mediated methylation of H3R17

The role of BACH1 in the process of vascular smooth muscle cell (VSMC) differentiation from human embryonic stem cells (hESCs) remains unknown. Here, we find that the loss of BACH1 in hESCs attenuates the expression of VSMC marker genes, whereas overexpression of BACH1 after mesoderm induction increases the expression of VSMC markers during invitro hESC-VSMC differentiation. Mechanistically, BACH1 binds directly to coactivator-associated arginine methyltransferase 1 (CARM1) during invitro hESC-VSMC differentiation, and this interaction is mediated by the BACH1 bZIP domain. BACH1 recruits CARM1 to VSMC marker gene promoters and promotes VSMC marker expression by increasing H3R17me2 modification, thus facilitating invitro VSMC differentiation from hESCs after the mesoderm induction. The increased expression of VSMC marker genes by BACH1 overexpression is partially abolished by inhibition of CARM1 or the H3R17me2 inhibitor TBBD in hESC-derived cells. These findings highlight the critical role of BACH1 in hESC differentiation into VSMCs by CARM1-mediated methylation of H3R17.

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.

Osteopontin is a key regulator of vascular smooth muscle cell proliferation in the outflow vein of arteriovenous fistulas.

Despite advances in the maintenance of arteriovenous fistulas (AVFs), the patency rates remain suboptimal. Most AVFs fail due to outflow vein stenosis; however, the underlying mechanism of AVF stenosis remains unclear. The present study aimed to identify key factors associated with AVF outflow stenosis. We obtained gene expression profiling data for the outflow vein of AVF from three Gene Expression Omnibus database datasets (GSE39488, GSE97377, and GSE116268) and analyzed the common differentially expressed genes (DEGs). We evaluated a common DEG in an aortocaval mouse model and the stenotic outflow veins of AVFs collected from patients. Furthermore, we isolated vascular smooth muscle cells (VSMCs) from the inferior vena cava (IVC) of wild-type (WT) and osteopontin (Opn)-knockout (KO) mice and assessed the proliferation of VSMCs following stimulation with platelet-derived growth factors (PDGFs). OPN was the only common upregulated DEG among all datasets. OPN was expressed in the medial layer of the outflow vein of AVF in aortocaval mouse models and co-stained with the VSMC marker (α-smooth muscle actin). OPN expression was markedly increased in the VSMCs of stenotic outflow veins of AVF collected from patients undergoing hemodialysis compared to presurgical veins acquired during AVF formation surgery. PDGF-induced VSMC proliferation was significantly increased in the VSMCs isolated from the IVC of WT mice but not in those isolated from the IVC of Opn-KO mice. OPN may be a key gene involved in VSMC proliferation in the AVF outflow veins and a therapeutic target to improve the AVF patency rate.

Apolipoprotein E (ApoE) Rescues the Contractile Smooth Muscle Cell Phenotype in Popliteal Artery Aneurysm Disease.

Popliteal artery aneurysm (PAA) is the most frequent peripheral aneurysm, primarily seen in male smokers with a prevalence below 1%. This exploratory study aims to shed light on cellular mechanisms involved in PAA progression. Sixteen human PAA and eight non-aneurysmatic popliteal artery samples, partially from the same patients, were analyzed by immunohistochemistry, fluorescence imaging, Affymetrix mRNA expression profiling, qPCR and OLink proteomics, and compared to atherosclerotic (n = 6) and abdominal aortic aneurysm (AAA) tissue (n = 19). Additionally, primary cell culture of PAA-derived vascular smooth muscle cells (VSMC) was established for modulation and growth analysis. Compared to non-aneurysmatic popliteal arteries, VSMCs lose the contractile phenotype and the cell proliferation rate increases significantly in PAA. Array analysis identified APOE higher expressed in PAA samples, co-localizing with VSMCs. APOE stimulation of primary human PAA VSMCs significantly reduced cell proliferation. Accordingly, contractile VSMC markers were significantly upregulated. A single case of osseous mechanically induced PAA with a non-diseased VSMC profile emphasizes these findings. Carefully concluded, PAA pathogenesis shows similar features to AAA, yet the mechanisms involved might differ. APOE is specifically higher expressed in PAA tissue and could be involved in VSMC phenotype rescue.

#4372 TYK2-INDUCED STAT ACTIVATION IS ESSENTIAL FOR VASCULAR PRO-CALCIFIC EFFECTS OF LEUKEMIA INHIBITORY FACTOR

Abstract Background and Aims Hyperphosphatemia in chronic kidney disease (CKD) is closely linked to medial vascular calcification. Phosphate is able to induce pro-inflammatory effects in vascular smooth muscle cells (VSMC), which could actively augment calcification processes. This study investigated the role of the IL-6 family member leukemia inhibitory factor (LIF) during vascular calcification. Method Experiments were performed in primary human aortic VSMCs, ex vivo mouse aortic rings and cholecalciferol treated mice, as well as serum samples from CKD patients and healthy controls. Results Phosphate exposure induced LIF release in VSMCs, while supplementation of LIF aggravated calcification and expression of pro-calcific markers in VSMCs. Silencing of LIF or addition of soluble LIF receptor (LIFR) as putative LIF antagonist ameliorated the pro-calcific effects of phosphate. Similarly, effects of phosphate or LIF were blunted by silencing of LIFR or its coreceptor GP130 in VSMCs. Mechanistically, LIF induced phosphorylation of TYK2, which was required for STAT1/3 activation. TYK2 overexpression augmented VSMC calcification, while silencing of either TYK2 or STAT1/3 ameliorated phosphate- and LIF-induced expression of pro-calcific markers. Pharmacological inhibition of TYK2 ameliorated calcification in VSMCs, mouse aortic rings and mice after cholecalciferol treatment. Furthermore, calcification was ameliorated in TYK2-deficient mouse aortic rings and mice after cholecalciferol overload. In a pilot study, LIFR was reduced in serum from patients with CKD and LIFR levels were associated with serum calcification propensity. Conclusion Vascular calcification is augmented by LIF, which identifies a crucial role of TYK2 in the pro-calcific reprogramming of VSMCs. Inhibition of this signalling axis might be able to reduce the burden of vascular calcification in CKD.

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.

ELUCIDATING LOCAL PHOSPHODIESTERASE 3A SIGNALING AS A BASIS FOR GAINING INSIGHT INTO HYPERTENSION-DRIVING MECHANISMS

Objective: Mutant PDE3A causes hypertension by driving mechanisms that increase peripheral vascular resistance. However, the media to lumen ratio of vessels in mutant PDE3A rat models varied between second-order mesenteric arteries and thoracic aorta. This observation may be related to alterations of the mutant PDE3A in its intracellular location, its hyperactivity and aberrant phosphorylation, which could lead to altered compartmentalized cAMP signaling. PDE3A hydrolyzes cAMP. Therefore, we hypothesized that alterations in compartmentalized cAMP signaling in vascular smooth muscle cells (VSMC) contributes to the hypertension. Design and method: Three different VSMC models were established: VSMCs from rat thoracic aorta, VSMCs from rat secondary mesenteric arteries and VSMCs expressing mutant PDE3A that were differentiated from human induced pluripotent stem cells. The expression levels of PDE3A and candidate genes were detected by immunofluorescence microscopy, Western blotting and qPCR based on results of RNA sequencing. cAMP and Ca2+ signaling pathways are being evaluated by FRET technology and Ca2+ imaging. Results: VSMCs differentiated from human induced pluripotent stem cells could detect abundant expression of vascular smooth muscle cell markers. Differential expression levels of PDE3A and the two screened candidate genes were found in different types of VSMCs. Conclusions: Elucidating differences in cAMP and Ca2+ cycling between different types of VSMCs yielded new insight into the mechanisms of hypertension. The mutant PDE3A signaling may be a starting point for an innovative, PDE3A compartment-specific pharmacological concept for the treatment of hypertension.

Single-cell RNA sequencing reveals the vascular smooth muscle cell phenotypic landscape in aortic aneurysm

Background and objectivesPhenotypic switching in vascular smooth muscle cells (VSMCs) has been linked to aortic aneurysm, but the phenotypic landscape in aortic aneurysm is poorly understood. The present study aimed to analyse the phenotypic landscape, phenotypic differentiation trajectory, and potential functions of various VSMCs phenotypes in aortic aneurysm.MethodsSingle-cell sequencing data of 12 aortic aneurysm samples and 5 normal aorta samples (obtained from GSE166676 and GSE155468) were integrated by the R package Harmony. VSMCs were identified according to the expression levels of ACTA2 and MYH11. VSMCs clustering was determined by the R package ‘Seurat’. Cell annotation was determined by the R package ‘singleR’ and background knowledge of VSMCs phenotypic switching. The secretion of collagen, proteinases, and chemokines by each VSMCs phenotype was assessed. Cell‒cell junctions and cell–matrix junctions were also scored by examining the expression of adhesion genes. Trajectory analysis was performed by the R package ‘Monocle2’. qPCR was used to quantify VSMCs markers. RNA fluorescence in situ hybridization (RNA FISH) was performed to determine the spatial localization of vital VSMCs phenotypes in aortic aneurysms.ResultsA total of 7150 VSMCs were categorize into 6 phenotypes: contractile VSMCs, fibroblast-like VSMCs, T-cell-like VSMCs, adipocyte-like VSMCs, macrophage-like VSMCs, and mesenchymal-like VSMCs. The proportions of T-cell-like VSMCs, adipocyte-like VSMCs, macrophage-like VSMCs, and mesenchymal-like VSMCs were significantly increased in aortic aneurysm. Fibroblast-like VSMCs secreted abundant amounts of collagens. T-cell-like VSMCs and macrophage-like VSMCs were characterized by high chemokine levels and proinflammatory effects. Adipocyte-like VSMCs and mesenchymal-like VSMCs were associated with high proteinase levels. RNA FISH validated the presence of T-cell-like VSMCs and macrophage-like VSMCs in the tunica media and the presence of mesenchymal-like VSMCs in the tunica media and tunica adventitia.ConclusionA variety of VSMCs phenotypes are involved in the formation of aortic aneurysm. T-cell-like VSMCs, macrophage-like VSMCs, and mesenchymal-like VSMCs play pivotal roles in this process.7meMEx4JhpJfRQ-F56EwAbVideo

Abstract 528: Endothelial Estrogen Receptor Alpha Deletion Promotes Abdominal Aortic Aneurysm In Male Mice

Introduction: Although men have a greater incidence of abdominal aortic aneurysm (AAA) compared with women, the role of sex-hormones in mediating these differences is not well understood. Given the known effect of estrogen receptor alpha (ERα) signaling in maintaining vascular wall homeostasis, the present study examines its role in the pathogenesis of AAA. Specifically, we scrutinize whether endothelial cell (EC) ERα protects against AAA via increased lysyl oxidase (LOX) activation and regulation of vascular smooth muscle cell (VSMC) phenotype. Methods: AAA was induced in male and female ECERα knockout (ECERαKO) mice and their littermates (n=6-11) by administering β-Aminopropionitrile (BAPN) via drinking water (1 mg/ml; 0.15 g/kg/d) from 3 days prior to angiotensin II (Ang II; 1 μg/kg/day) infusion for 2 weeks. The development of AAA was assessed by ultrasound measurement of aortic luminal dilation and histology. Relevant molecular mechanisms were examined in abdominal aorta and in human aortic endothelial cells (HAECs). Results: BAPN Ang II resulted in an increased incidence of AAA in male ECERαKO mice (72%), whereas female ECERαKO mice did not develop aneurysms. No difference in AAA incidence was observed between male and female littermate control mice (12.5%). Elastin fragmentation was significantly higher only in male ECERαKO mice compared to littermates, while female ECERαKO and their littermates had comparable elastin fragmentation. Ang II receptor type 1a mRNA expression was higher in male ECERαKO, whereas female ECERαKO mice had higher Ang II receptor type 1b mRNA expression. Gene expression of smooth muscle alpha-actin and smooth muscle 22 (contractile VSMC markers) was significantly higher in female aortas compared to males irrespective of genotype, whereas, osteopontin, synthetic VSMC marker was only upregulated in male ECERαKO. HAECs treated with estradiol (10 nM, 24 h) significantly increased LOX activity. Conclusions: Our findings identify a critical role of endothelial ERα in protection against AAA in male mice likely through increased LOX activity and maintenance of VSMC phenotype. Further studies are needed to delineate the crosstalk between endothelial ERα and VSMCs, and their impact in the pathogenesis of AAA.

Abstract 212: Transcriptional Profiling Of Vascular Smooth Muscle Cell-foam Cells In Atherosclerotic Lesions

Background: One of the hallmarks of atherosclerosis progression is the uptake of oxidized lipids by cells to become Foam/Foamy cells - the most prominent being vascular smooth muscle cells (VSMC)-foam cells. Although studies have identified the ability of VSMC to become VSMC-foam cells, their transcriptomic profile and the phenotypic modulations which distinguish them from contractile VSMC remain poorly defined and understood. Hypothesis: We hypothesize that VSMC-foamy cells differ from contractile VSMC not only by modulation of genes shared by both cell types, but also by the expression of a unique set of genes which contribute to their formation and maintenance within atherosclerotic lesions. Method: To address this, we performed bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing mice models on high fat diet. VSMC and VSMC-foam cells were isolated and sequenced at specific time points reflective of late and advanced atherosclerosis disease progression. Computational analysis enabled us identify differentially expressed genes, biological pathways and molecular functions which distinguished VSMC from VSMC-foam cells. Result and Conclusion: Our computational analysis identified differential genes and pathways unique to VSMC-foam cells which might be associated with disease progression. From our analysis, VSMC showed a lower expression of contractile VSMC marker genes and a higher expression of genes in synthetic/intermediary multipotent state, we term “SEM” state. Our study also showed that VSMC-foam cells also expressed unique genes which distinguished them from contractile VSMC. These genes not only serve as potential genetic markers, but might further explain the nuclear composition, as well as the inflammatory and metabolic profile of VSMC-foam cells. Defining and understanding the molecular mechanisms and drivers which promote VSMC-foam cell formation and maintenance in atherosclerosis, would aid in our understanding of the disease with potentials for more effective therapeutic solutions.

Abstract 217: Protease Activated Receptor 2 Is Critical For Vascular Smooth Muscle Cell Transition To A Macrophage-like State

Objective: Recent lineage tracing studies have shown vascular smooth muscle cells (VSMCs) develop macrophage-like characteristics (MLCs) in late-stage atherosclerosis resulting in accelerated inflammation and disease severity. Our objective was to determine if the inflammatory receptor, protease activated receptor 2 (PAR2), accelerates VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Our lab demonstrated Par2 -/- mice have decreased atherosclerotic lesion area versus proficient controls via downregulation of VSMC migration and cytokine release. To examine the role of PAR2 in VSMC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10 ug/mL methyl- β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers (α - actin and myosin heavy chain) and upregulated macrophage markers (CD68 and Mac-2), Par2 -/- VSMCs remained stable. Looking at macrophage characteristics, Par2 -/- VSMCs phagocytized significantly less latex beads than Par2 +/+ VSMCs. Analysis of a hybrid mouse diversity panel (HMDP), conducted on 101 strains of mice with induced atherosclerosis, determined PAR2 was significantly correlated with the MLC super-gene Krüppel-like factor 4 (KLF4), which was found to have lower genetic expression in Par2 deficient VSMCs. Previous studies have demonstrated Par2 mRNA is bound and stabilized by the RNA binding protein human antigen R (HuR). Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression and activity, suggesting a role of HuR in PAR2 mRNA stability. Par2 flox/flox mice bred with transgelin (SM22-alpha) Cre and low-density lipoprotein receptor deficient mice l have been establishing in our lab to investigate the effects of cell-specific deletions of PAR2 in atherosclerosis. VSMC-specific Par2 deficiency blunts the formation of atherosclerosis versus the Cre + littermates. Conclusions: These results suggest that VSMC-specific PAR2 activation mediates the dedifferentiation of VSMCs via upregulation of KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. Future studies will continue to study VSMC dedifferentiation utilizing tagged lineage-tracing mouse models and various in vitro techniques.