Contractile Vascular Smooth Muscle Cells Research Articles

Enhanced Expression of miR-20a Driven by Nanog Exacerbated the Degradation of Extracellular Matrix in Thoracic Aortic Dissection

Thoracic aortic dissection (TAD) is a life-threatening vascular disease manifested as intramural bleeding in the medial layers of the thoracic aorta. The key histopathologic feature of TAD is medial degeneration, characterized by depletion of vascular smooth muscle cells (VSMCs) and degradation of extracellular matrix (ECM). MicroRNA, as essential epigenetic regulators, can inhibit the protein expression of target genes without modifying the sequences. This study aimed to elucidate the role and underlying mechanism of miR-20a, a member of the miR-17-92 cluster, in regulating ECM degradation during the pathogenesis of TAD. The expression of the miR-17-92 cluster was significantly increased in synthetic VSMCs derived from TAD lesions compared to contractile VSMCs isolated from normal thoracic aortas. Notably, the expression of miR-20a was increased in VSMCs in response to serum exposure and various stimuli. In TAD lesions, the expression of miR-20a was significantly negatively correlated with that of elastin. Elevated expression of miR-20a was also observed in thoracic aortas of TAD mice induced by β-aminopropionitrile fumarate and angiotensin II. Overexpression of miR-20a via mimic transfection enhanced the growth and invasive capabilities of VSMCs, with no significant impact on their migratory activity or the expression of phenotypic markers (α-SMA, SM22, and OPN). Silencing of miR-20a with inhibitor transfection mitigated the hyperactivation of MMP2 in VSMCs stimulated by PDGF-bb, as evidenced by reduced levels of active-MMP2 and increased levels of pro-MMP2. Subsequently, TIMP2 was identified as a novel target gene of miR-20a. The role of miR-20a in promoting the activation of MMP2 was mediated by the suppression of TIMP2 expression in VSMCs. In addition, the elevated expression of miR-20a was found to be directly driven by Nanog in VSMCs. Collectively, these findings indicate that miR-20a plays a crucial role in maintaining the homeostasis of the thoracic aortic wall during TAD pathogenesis and may represent a potential therapeutic target for TAD.

Vascular Smooth Muscle Cell Mechanical Stretch Modulates Tissue Transglutaminase Activity and Cytoskeletal Dynamics

Vascular smooth muscle cells (VSMCs) are exposed to a wide range of mechanical stimuli in both physiological and pathological conditions. These mechanical stimuli are converted into biochemical signals that regulate VSMC contractility and cytoskeletal structures in a process known as mechanotransduction. Thus, the ability of VSMCs to sense and respond to mechanical signals is essential for proper regulation of their structural characteristics and the modulation of vascular tone. We had previously shown that increased tissue transglutaminase 2 (TG2) activity modulates actin dynamics via alterations of the phosphorylation state of cofilin leading to VSMC stiffening and remodeling of the vascular wall. However, whether mechanical forces act through TG2 to modulate VSMC stiffness remains unknown. Herein, we tested the hypothesis that mechanical stretch increases VSMC stiffness by increasing TG2 activity, reducing cofilin activity, and increasing actin cytoskeletal stress fiber formation. To this end, immortalized human aortic vascular smooth muscle cells (HAoSMCs) were cultured and seeded on Uniflex culture plates. HAoSMCs were left unstretched or exposed to cyclic uniaxial stretch using a Flexcell tension system with 11% elongation at 1 Hz for 6 hrs. HAoSMCs were treated without or with the nitric oxide (NO) donor s-nitrosoglutathione (400μmol/L) or cystamine (200μmol/L) under stretched and unstretched conditions, as NO and cystamine are known inhibitors of TG2. All differences reported herein are significant at p<0.05. Our results show that cyclic uniaxial stretch for 6 hrs increases VSMC TG2 content and activity, actin stress fibers (polymerization), and phospho-cofilin/cofilin ratios (cofilin inactivation), relative to the unstretched condition. Notably, the increase in TG2 activity and F-actin content occurring in response to stretch was inhibited by the NO donor or cystamine. As it has been previously shown that cellular stiffness is associated with stress fiber formation, this work suggests that stretch-induced cellular stiffening is potentially mediated by TG2 activity in VSMC. National Institutes of Health grant: R01HL153264 to LM-L and JP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel Role of YAP1 in Regulating Blood Pressure and Vascular Tone

Introduction: Hypertension is a major risk factor for occlusive vascular diseases, myocardial infarction, heart failure, stroke, and chronic kidney disease. In the U.S., ~30% of adults suffer from hypertension. Merely half of the individuals with hypertension have their blood pressure under control, despite the wide range of antihypertensive medications available, indicating an urgent need for a better understanding of the underlying causes of hypertension to identify novel therapeutic strategies for its treatment and prevention. Recent genome-wide association study (GWAS) has identified an unexpected association between a loss-of-function SNP in the YAP1 gene locus and decreased blood pressure (BP). Moreover, de novo analysis of publicly available RNAseq data revealed that the vascular tissues of hypertensive mice treated with Ang-II exhibit increased YAP1 expression. Suggesting a novel role of YAP1 in blood pressure regulation. However, whether Vascular smooth muscle cell (VSMC) expressed YAP1 has a specific role in VSMC contraction and BP regulation remains completely unknown. Therefore, we hypothesized that YAP1 could play a key role in regulating blood pressure. Methodology: We have generated a novel inducible SM-specific- Yap1 Knockout ( YAP1 iKO) mouse model and employed a pharmacological inhibitor of YAP1, Verteporfin, to delineate the functional role of VSMC-expressed YAP1 in hypertension in regulating vascular tone and BP. Vascular reactivity experiments were performed using wire myography. BP was measured in ambulant mice via radiotelemetry. In vitro gain- and loss-of-function studies utilizing human coronary artery SMCs were conducted to examine the impact of YAP1 on contractility signaling components using western blotting. Results: Consistent with GWAS and RNAseq data, we found that YAP1 is upregulated in vascular tissues of spontaneously hypertensive rats (SHRs). Using aortic rings from WT mice, we discovered that Verteporfin significantly reduced vasoconstrictive responses to Phenylephrine or Serotonin but did not alter endothelium-dependent vasorelaxant responses to Acetylcholine, suggesting a specific role of YAP1 in VSMCs. Consistently, YAP1 iKO exhibited attenuated responses to vasoconstrictors in isolated conduit and resistance vessels. Importantly, radiotelemetry studies demonstrated for the first time that YAP1 iKO mice exhibit a hypotensive phenotype. Mechanistically, YAP1 gain- and loss-of-function studies in human VSMCs showed that YAP1 activates multiple signaling pathways that play a key role in VSMC contractility including RhoA/ROCK1/actin polymerization and PKC/ERK signaling pathways. Summary/Conclusion: This is the first study to demonstrate that inhibition of SM-specific-Yap1 expression regulates BP and vascular tone, likely via the regulation of contractile machinery components. Together, our data suggest that YAP1 is a promising novel therapeutic target for ameliorating hypertension. This work is supported by a grant from National Heart, Lung, and Blood Institute (R00HL153896). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Primary Mouse Aortic Smooth Muscle Cells Exhibit Region- and Sex-Dependent Biological Responses In Vitro.

Despite advancements in elucidating biological mechanisms of cardiovascular remodeling, cardiovascular disease (CVD) remains the leading cause of death worldwide. When stratified by sex, clear differences in CVD prevalence and mortality between males and females emerge. Regional differences in phenotype and biological response of cardiovascular cells are important for localizing the initiation and progression of CVD. Thus, to better understand region and sex differences in CVD presentation, we have focused on characterizing in vitro behaviors of primary vascular smooth muscle cells (VSMCs) from the thoracic and abdominal aorta of male and female mice. VSMC contractility was assessed by traction force microscopy (TFM; single cell) and collagen gel contraction (collective) with and without stimulation by transforming growth factor-beta 1 (TGF-β1) and cell proliferation was assessed by a colorimetric metabolic assay (MTT). Gene expression and TFM analysis revealed region- and sex-dependent behaviors, whereas collagen gel contraction was consistent across sex and aortic region under baseline conditions. Thoracic VSMCs showed a sex-dependent sensitivity to TGF-β1-induced collagen gel contraction (female > male; p = 0.025) and a sex-dependent proliferative response (female > male; p < 0.001) that was not apparent in abdominal VSMCs. Although primary VSMCs exhibit intrinsic region and sex differences in biological responses that may be relevant for CVD presentation, several factors-such as inflammation and sex hormones-were not included in this study. Such factors should be included in future studies of in vitro mechanobiological responses relevant to CVD differences in males and females.

Small nucleolar RNA host gene 18 controls vascular smooth muscle cell contractile phenotype and neointimal hyperplasia.

Long non-coding RNA (LncRNA) small nucleolar RNA host gene 18 (SNHG18) has been widely implicated in cancers. However, little is known about its functional involvement in vascular diseases. Herein, we attempted to explore a role for SNHG18 in modulating vascular smooth muscle cell (VSMC) contractile phenotype and injury-induced neointima formation. Analysis of single-cell RNA sequencing and transcriptomic datasets showed decreased levels of SNHG18 in injured and atherosclerotic murine and human arteries, which is positively associated with VSMC contractile genes. SNHG18 was upregulated in VSMCs by TGFβ1 through transcription factors Sp1 and SMAD3. SNHG18 gene gain/loss-of-function studies revealed that VSMC contractile phenotype was positively regulated by SNHG18. Mechanistic studies showed that SNHG18 promotes a contractile VSMC phenotype by up-regulating miR-22-3p. SNHG18 up-regulates miR-22 biogenesis and miR-22-3p production by competitive binding with the A-to-I RNA editing enzyme, adenosine deaminase acting on RNA-2 (ADAR2). Surprisingly, we observed that ADAR2 inhibited miR-22 biogenesis not through increasing A-to-I editing within primary miR-22, but by interfering with the binding of microprocessor complex subunit DGCR8 to primary miR-22. Importantly, perivascular SNHG18 overexpression in the injured vessels dramatically up-regulated the expression levels of miR-22-3p and VSMC contractile genes, and prevented injury-induced neointimal hyperplasia. Such modulatory effects were reverted by miR-22-3p inhibition in the injured arteries. Finally, we observed a similar regulator role for SNHG18 in human VSMCs and a decreased expression level of both SNHG18 and miR-22-3p in diseased human arteries; and we found that the expression level of SNHG18 was positively associated with that of miR-22-3p in both healthy and diseased human arteries. We demonstrate that SNHG18 is a novel regulator in governing VSMC contractile phenotype and preventing injury-induced neointimal hyperplasia. Our findings have important implications for therapeutic targeting snhg18/miR-22-3p signalling in vascular diseases.

Arterial Stiffness: From Basic Primers to Integrative Physiology.

The elastic properties of conductance arteries are one of the most important hemodynamic functions in the body, and data continue to emerge regarding the importance of their dysfunction in vascular aging and a range of cardiovascular diseases. Here, we provide new insight into the integrative physiology of arterial stiffening and its clinical consequence. We also comprehensively review progress made on pathways/molecules that appear today as important basic determinants of arterial stiffness, particularly those mediating the vascular smooth muscle cell (VSMC) contractility, plasticity and stiffness. We focus on membrane and nuclear mechanotransduction, clearance function of the vascular wall, phenotypic switching of VSMCs, immunoinflammatory stimuli and epigenetic mechanisms. Finally, we discuss the most important advances of the latest clinical studies that revisit the classical therapeutic concepts of arterial stiffness and lead to a patient-by-patient strategy according to cardiovascular risk exposure and underlying disease.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

Phenotypic switching of vascular smooth muscle cells in atherosclerosis, hypertension, and aortic dissection.

Vascular smooth muscle cells (VSMCs) play a critical role in regulating vasotone, and their phenotypic plasticity is a key contributor to the pathogenesis of various vascular diseases. Two main VSMC phenotypes have been well described: contractile and synthetic. Contractile VSMCs are typically found in the tunica media of the vessel wall, and are responsible for regulating vascular tone and diameter. Synthetic VSMCs, on the other hand, are typically found in the tunica intima and adventitia, and are involved in vascular repair and remodeling. Switching between contractile and synthetic phenotypes occurs in response to various insults and stimuli, such as injury or inflammation, and this allows VSMCs to adapt to changing environmental cues and regulate vascular tone, growth, and repair. Furthermore, VSMCs can also switch to osteoblast-like and chondrocyte-like cell phenotypes, which may contribute to vascular calcification and other pathological processes like the formation of atherosclerotic plaques. This provides discusses the mechanisms that regulate VSMC phenotypic switching and its role in the development of vascular diseases. A better understanding of these processes is essential for the development of effective diagnostic and therapeutic strategies.

Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity.

Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes.

Vascular Inflammation and Smooth Muscle Contractility: The Role of Nox1-Derived Superoxide and LRRC8 Anion Channels.

Vascular inflammation underlies the development of hypertension, and the mechanisms by which it increases blood pressure remain the topic of intense investigation. Proinflammatory factors including glucose, salt, vasoconstrictors, cytokines, wall stress, and growth factors enhance contractility and impair relaxation of vascular smooth muscle cells. These pathways share a dependence upon redox signaling, and excessive activation promotes oxidative stress that promotes vascular aging. Vascular smooth muscle cell phenotypic switching and migration into the intima contribute to atherosclerosis, while hypercontractility increases systemic vascular resistance and vasospasm that can trigger ischemia. Here, we review factors that drive the initiation and progression of this vasculopathy in vascular smooth muscle cells. Emphasis is placed on the contribution of reactive oxygen species generated by the Nox1 NADPH oxidase which produces extracellular superoxide (O2•-). The mechanisms of O2•- signaling remain poorly defined, but recent evidence demonstrates physical association of Nox1 with leucine-rich repeat containing 8 family volume-sensitive anion channels. These may provide a pathway for influx of O2•- to the cytoplasm, creating an oxidized cytoplasmic nanodomain where redox-based signals can affect both cytoskeletal structure and vasomotor function. Understanding the mechanistic links between inflammation, O2•- and vascular smooth muscle cell contractility may facilitate targeting of anti-inflammatory therapy in hypertension.

Interstitial Fluid Shear Stress Induces the Synthetic Phenotype Switching of VSMCs to Release Pro-calcified Extracellular Vesicles via EGFR-MAPK-KLF5 Pathway.

Phenotypic switching (from contractile to synthetic) of vascular smooth muscle cells (VSMCs) is essential in the progression of atherosclerosis. The damaged endothelium in the atherosclerotic artery exposes VSMCs to increased interstitial fluid shear stress (IFSS). However, the precise mechanisms by which increased IFSS influences VSMCs phenotypic switching are unrevealed. Here, we employed advanced numerical simulations to calculate IFSS values accurately based on parameters acquired from patient samples. We then carefully investigated the phenotypic switching and extracellular vesicles (EVs) secretion of VSMCs under various IFSS conditions. By employing a comprehensive set of approaches, we found that VSMCs exhibited synthetic phenotype upon atherosclerotic IFSS. This synthetic phenotype is the upstream regulator for the enhanced secretion of pro-calcified EVs. Mechanistically, as a mechanotransducer, the epidermal growth factor receptor (EGFR) initiates the flow-based mechanical cues to MAPK signaling pathway, facilitating the nuclear accumulation of the transcription factor krüppel-like factor 5 (KLF5). Furthermore, pharmacological inhibiting either EGFR or MAPK signaling pathway blocks the nuclear accumulation of KLF5 and finally results in the maintenance of contractile VSMCs even under increased IFSS stimulation. Collectively, targeting this signaling pathway holds potential as a novel therapeutic strategy to inhibit VSMCs phenotypic switching and mitigate the progression of atherosclerosis.

STELLATE GANGLION BLOCK REVERSES PHSML-INDUCED VASCULAR HYPOREACTIVITY THROUGH INHIBITING AUTOPHAGY-MEDIATED PHENOTYPIC TRANSFORMATION OF VSMCs.

Posthemorrhagic shock mesenteric lymph (PHSML) return-contributed excessive autophagy of vascular smooth muscle cells (VSMCs) is involved in vascular hyporeactivity, which is inhibited by stellate ganglion block (SGB) treatment. The contractile phenotype of VSMCs transforms into a synthetic phenotype after stimulation with excessive autophagy. Therefore, we hypothesized that SGB ameliorates PHSML-induced vascular hyporeactivity by inhibiting autophagy-mediated phenotypic transformation of VSMCs. To substantiate this hypothesis, a hemorrhagic shock model in conscious rats was used to observe the effects of SGB intervention or intravenous infusion of the autophagy inhibitor 3-methyladenine (3-MA) on intestinal blood flow and the expression of autophagy- and phenotype-defining proteins in mesenteric secondary artery tissues. We also investigated the effects of intraperitoneal administration of PHSML intravenous infusion and the autophagy agonist rapamycin (RAPA) on the beneficial effect of SGB. The results showed that hemorrhagic shock decreased intestinal blood flow and enhanced the expression of LC3 II/I, Beclin 1, and matrix metalloproteinase 2, which were reversed by SGB or 3-MA treatment. In contrast, RAPA and PHSML administration abolished the beneficial effects of SGB. Furthermore, the effects of PHSML or PHSML obtained from rats treated with SGB (PHSML-SGB) on cellular contractility, autophagy, and VSMC phenotype were explored. Meanwhile, the effects of 3-MA on PHSML and RAPA on PHSML-SGB were observed. The results showed that PHSML, but not PHSML-SGB, incubation decreased VSMC contractility and induced autophagy activation and phenotype transformation. Importantly, 3-MA administration reversed the adverse effects of PHSML, and RAPA treatment attenuated the effects of PHSML-SGB incubation on VSMCs. Taken together, the protective effect of SGB on vascular reactivity is achieved by inhibiting excessive autophagy-mediated phenotypic transformation of VSMCs to maintain their contractile phenotype.

Olfactory receptor 2 signaling drives experimental abdominal aortic aneurysm formation

Abstract Background Abdominal aortic aneurysms (AAA) are defined as enlargement above 3cm or 50% of its original diameter. Therapeutic options are limited to surgical and endovascular treatment, while no pharmacological interventions are available. Chronic inflammation of the vessel wall and in particularly macrophage infiltration has been associated with disease progression, leading to extensive tissue remodeling, yet the underlying mechanisms are incompletely understood. A recent study has shown extra-nasal expression of the olfactory receptor 2 (OLFR2) on vascular macrophages, in which activation induced a pro-atherogenic response. The role of macrophage OLFR2 signaling in AAA, however, is unknown. Methods &amp; Results To determine whether the OLFR2 ortholog OR6A2 is expressed in human AAA, we performed in silico micro-array analysis of human AAA tissue. OR6A2 was shown to be specifically upregulated in AAA tissue with thrombus formation compared to healthy control tissue. We confirmed OR6A2 expression on macrophages by immunofluorescence. To determine OLFR2 expression among vascular immune cells in mice, we induced AAA formation by porcine pancreas (PPE) infusion into the infrarenal aorta of C57BL6/J (WT) mice and performed spectral cytometry utilizing a 26-marker panel at baseline, day 7 and day 28. OLFR2 expression peaked at day 7 and decreased to baseline levels at day 28 particularly on pro-inflammatory monocytes, mixed resident / migratory-like macrophages and pro-inflammatory macrophages, while OLFR2 was not detectable on tissue resident-like macrophages. To assess a functional role of OLFR2 in AAA formation, we induced AAA in OLFR2-deficient (KO) mice and WT controls by PPE. Ultrasound analysis of the abdominal aorta revealed protection from AAA formation in KO mice. Histological analysis showed significantly reduced macrophage while increased smooth muscle cell content in aortic sections of KO mice compared to WT controls. This was accompanied by higher collagen content in KO mice. Additionally, plasma levels of the pro-inflammatory cytokines TNF-alpha and Interferon-gamma were significantly reduced in KO mice 28 days post PPE. Bulk transcriptome analysis corroborated these findings and indicated increased pathways associated with vascular smooth muscle cell contractility while decreased leukocyte activation in KO aortas. Conclusion We show that OLFR2 deficiency in mice attenuates abdominal aortic aneurysm development, aortic remodeling and pro-inflammatory response. OLFR2 might thus present a novel target for drug-based AAA therapies.

Abstract 016: GRN Gene Mutation Leads To Vascular Abnormalities Via Mitochondrial Dysfunction And Oxidative Stress

Background: Cardiovascular disease (CVD) is the leading cause of death worldwide. Loss of contractility of vascular smooth muscle cells (VSMC) is a major factor of CVD. The GRN gene encodes the glycoprotein progranulin (PGRN). Ubiquitously expressed, PGRN is implicated in anti-inflammation and cell proliferation. However, the role of PGRN in CVD remains to be elucidated. Herein, we tested the hypothesis that PGRN influences VSMC contraction via regulating mitochondria quality. Methods: We used aortae from wildtype (PGRN +/+ ) and PGRN global knockout (PGRN -/- ) mice to study vascular contraction via wire myograph and isolate primary aVSMCs to elucidate mechanisms. Results: We observed a significantly suppressed contractile profile in PGRN -/- aVSMC characterized by reduced α smooth muscle actin expression and impaired VSMC contractility, analyzed by collagen disc assay. PGRN -/- aortae demonstrated diminished vascular contractility to KCl (maximal response in mN, PGRN +/+ 5.3±0.3 vs PGRN -/- 4.0±0.4*, *P&lt;0.05) and thromboxane A2 analog, U46619 (maximal response in mN, PGRN +/+ 6.0±0.5 vs PGRN -/- 4.7±0.3*, *P&lt;0.05). RNA-sequencing in aortae revealed a suppression in oxidative phosphorylation in PGRN -/- . Furthermore, PGRN -/- VSMC and aortae displayed a suppressed mitochondrial oxygen consumption rate (OCR) analyzed by Seahorse and Oroboros followed by attenuated ATP levels and elevated mitochondrial oxidative stress. No difference was observed for mitochondria DNA number or biogenesis. To rescue the PGRN levels, we incubated aortae from PGRN -/- mice with lentivirus (LV) encoding PGRN for 48h, which elevated the vascular contractility to KCl (maximal response in mN, PGRN -/- 4.8 ± 0.4 vs PGRN -/-LV 7.1 ± 0.3*, *P&lt;0.05) and U46619 (maximal response in mN, PGRN -/- 8.0 ± 0.2 vs PGRN -/-LV 16.0 ± 0.2*, *P&lt;0.05), increased OCR, and controlled the oxidative stress, whereas inhibiting mitochondria with rotenone prevented the LV effects. Conclusion: Collectively, we demonstrated that PGRN is essential to maintain vascular contractility via regulating mitochondrial function. Our results show that loss-of-function of PGRN is a risk factor for developing CVD and places PGRN as a potential alternative therapeutic target for high-risk CVD populations.

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.

THE INHIBITORY EFFECTS OF VALPROIC ACID ON RHOA-MEDIATED VASCULAR SMOOTH MUSCLE CELL CONTRACTION

Objective: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. Growing evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of VPA on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which VPA inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Design and method: We performed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), RhoA activity assay, transfection of constitutively active (CA)-RhoA gene, and western blot analyses. We also conducted ex vivo studies using isolated rat aortas. Results: VPA inhibited phenylephrine (PE)-induced phosphorylation of myosin light chain (MLC) at Ser19 (p-MLC-Ser19) in VSMCs. VPA did not alter expressions of MLC kinase and Rho-associated kinase 1, the upstream kinases responsible for MLC phosphorylation. In addition, VPA did not affect phosphorylation of myosin phosphatase target subunit 1, a regulatory subunit of myosin phosphatase. VPA decreased RhoA mRNA expression and its protein levels in a time- and dose-dependent manner, and as expected, VPA decreased amount of active GTP-bound RhoA in a dose-dependent manner. Ectopic expression of CA-RhoA gene significantly reversed VPA-inhibited p-MLC-Ser19 and p-CPI-17-Thr38. Finally, VPA significantly attenuated PE-induced vessel contraction in endothelium-deprived rat aortas, and similar to the in vitro results, VPA decreased RhoA expression and levels of p-MLC-Ser19 and p-CPI-17-Thr38 in rat aortas. Conclusions: We demonstrate that VPA mitigates PE-induced VSMC contractility and vessel contraction by decreasing expressions of RhoA and p-MLC-Ser19 in rat VSMCs and aortas. These results suggest that VPA may be useful in the treatment of vasospastic diseases including coronary artery vasospasm and post-subarachnoid hemorrhage cerebral vasospasm.

INKILN is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.

Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1β-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.

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

Apelin Regulation of K-Cl Cotransport in Vascular Smooth Muscle Cells as a Potential Target for Cardiovascular Disease

Background/Aims: Apelin and its signaling through the G-protein coupled receptor (APJ, gene symbol APLNR) regulate cardiovascular function via two mechanisms: 1) By promoting nitric oxide (NO)-mediated vasodilation, impaired by oxidized low-density lipoproteins (oxLDL); and 2) By inducing cell proliferation via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/Akt pathway (PI3K/Akt) and mitogen activated protein kinase (MAPK) pathways. The potassium chloride cotransporter (KCC1,3,4; SLC12A4,6,7) controls cell volume, and regulates cardiovascular function through proliferation, migration, and blood pressure control. Importantly, KCC regulatory mechanisms and apelin/APJ signaling pathways overlap placing KCC as a potential target for apelin/APJ to elicit its cardioprotective effects. Therefore, apelin’s action on KCC activity was examined in contractile and synthetic rat aortic vascular smooth muscle cells (VSMCs). Methods: KCC activity was measured by atomic absorption spectrophotometry in chloride (Cl-) and Cl--free medium with sulfamate (Sf-) as Cl- replacement, and with rubidium (Rb+) as a potassium (K+) congener. The calculated difference between Rb+ transport in the presence of chloride (Cl-) and sulfamate (Sf-) is the Cl--dependent Rb+ influx (i.e., K-Cl cotransport activity). Apelin-13 (1 µM) was added either during flux (acute effect) and/or in the growth media (chronic effect) based on the experimental goals. KCC activity was characterized with respect to the VSMC phenotypes, in the presence or absence of apelin and corresponding inhibitors of the signaling pathways, oxLDL, and as a function of various physiological factors described below. Results: The APJ receptor was expressed in both contractile and synthetic VSMC phenotypes, the former also possessing the soluble guanylyl cyclase-coupled protein kinase G (PKG) receptor, critical for NO-mediated signaling. In general, KCC activity was higher in synthetic vs. contractile VSMCs, consistent with enhanced migration and proliferation in the former. In addition, apelin-mediated activation of KCC was modulated by extracellular sodium [Na+]o, osmolality, length of apelin treatment (acute or chronic) and VSMC phenotype (contractile vs synthetic). Based on selective inhibitors, apelin activated KCC through the (NO)/soluble guanylate cyclase (sGC)/protein kinase G (PKG) (NO/sGC/PKG)-, PI3K/Akt- and MAPK-dependent pathway(s). Furthermore, apelin rescued the inhibition of KCC by oxLDL. Altogether, results suggest apelin/APJ as an important modulator of KCC activity to sustain cell volume regulation and cardiovascular function. More recently, the apelinergic system has been proposed as a novel target for the treatment of Corona virus disease 2019 (COVID-19) and, given the significant overlap between the regulatory mechanisms of this system and KCC, and their role in cardiovascular disease (CVD), this study opens new avenues to identify potential targets for diverse implementation strategies. Conclusion: A better understanding of apelin effects on KCC will help design a novel therapeutic approach to treat atherosclerosis-linked cardiovascular diseases, including COVID-19-associated mortality.

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.