Aortic Vascular Smooth Muscle Research Articles

Empagliflozin Attenuates Neointima Formation After Arterial Injury and Inhibits Smooth Muscle Cell Proliferation and Migration by Suppressing Platelet-Derived Growth Factor-Related Signaling.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events. However, the precise mechanisms beyond glycemic control are not fully understood. The objective of this study was to determine the role of PDGF (platelet-derived growth factor)-related signaling in empagliflozin-mediated inhibition of neointima formation. Adult male nondiabetic Wistar rats were subjected to carotid artery balloon injury. Empagliflozin (30 mg/kg per day) was administered by oral gavage for 18 days beginning 4 days before surgery. The invitro effects of empagliflozin on rat aortic vascular smooth muscle cell (VSMC) proliferation and migration were also determined. Empagliflozin attenuated balloon injury-induced neointima formation in carotid arteries. In VSMCs, empagliflozin attenuated PDGF-BB-induced proliferation and migration. Moreover, empagliflozin-treated VSMCs did not undergo apoptosis or cytotoxic death. Empagliflozin suppressed PDGF-related signaling, including phosphorylation of PDGF receptor β, Akt, and STAT3 (signal transducer and activator of transcription 3). Overactivation of PDGF signaling attenuated empagliflozin-mediated inhibition of VSMC function. SGLT2 mRNA levels in rat VSMCs were undetectable, and SGLT2 silencing did not alter the empagliflozin-mediated effects, supporting the SGLT2-independent effects of empagliflozin on VSMC. This study highlights the crucial role of suppressing PDGF-related signaling in mediating the beneficial effects of empagliflozin on neointima formation and VSMC function, which are independent of SGLT2 and glycemic control. Our study provides a novel mechanistic aspect of empagliflozin for the prevention of vascular stenosis disorders.

Hsa_circ_0007765 Promotes Platelet-Derived Growth Factor-BB-Induced Proliferation and Migration of Human Aortic Vascular Smooth Muscle Cells in Atherosclerosis.

Humanaorticvascularsmoothmusclecells (HA-VSMCs) play vital roles in the pathogenesis ofvasculardiseases, including Atherosclerosis (AS). Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs.Therefore, this study aimed to explore the role and mechanism of hsa_circRNA_102353 (circ_0007765) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs. Circ_0007765, microRNA-654-3p (miR-654-3p), and Fibroblast Growth Factor Receptor Substrate 2 (FRS2) expression were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferative ability, invasion, and migration were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and wound healing assays. CyclinD1, MMP2, and FRS2 protein levels were assessed using a Western blot assay. Binding between miR-654-3p and circ_0007765 or FRS2 was predicted by Circinteractome or TargetScan, and verified using dual-luciferase reporter and RNA pull-down assays. PDGF-BB induced HA-VSMC proliferation, invasion, and migration. Circ_0007765 and FRS2 expression levels were increased in PDGF-BB-treated HA-VSMCs, and the miR-654-3p level was reduced. Moreover, circ_0007765 absence hindered PDGF-BB-induced HA-VSMC proliferation, invasion, and migration in vitro. At the molecular level, circ_0007765 increased FRS2 expression by acting as a sponge for miR-654-3p. Our findings revealed that circ_0007765 boosted PDGF-BB-induced HA-VSMC proliferation and migration through elevating FRS2 expression via adsorbing miR-654-3p, providing a feasible therapeutic strategy for AS.

Chungtaejeon (CTJ) inhibits adhesion and migration of VSMC through cytoskeletal remodeling pathway

IntroductionVascular remodeling is crucial for the progression of vascular disease such as atherosclerosis. We utilize the in vitro experimental model of atherosclerosis to elucidate the activity of Chungtaejeon (CTJ), a Korean fermented tea on adhesion and migration of human aortic vascular smooth muscle cells (HASMC). Materials and methodsVarious in vitro assays such as cell viability, cell adhesion, Western blot, immunofluorescence, were carried out on HASMC to explore pathway associated with cytoskeletal remodeling during the progression of atherosclerosis. ResultsIn result, CTJ significantly inhibited adhesion of HASMC as revealed by collagen assay. Similarly, CTJ inhibited the β1-integrin protein expression as well as FAK phosphorylation. Treatment of CTJ also inhibited stress fiber formation. Likewise, adherence of cells on collagen optimally increased the expression of both RhoA and Cdc42, however, treatment of CTJ dose dependently decreased their expression. The lysophosphatidic acid stimulation of HASMC rapidly increased the level of phosphorylated forms of MLC20 within 15 min, followed by an extended level of MLC20 phosphorylation. The treatment of CTJ at a dose of 50, 100 and 250 μg/ml remarkably reduced the diphosphorylated form while decreased the level of monophosphorylated form of MLC20. ConclusionsOur results suggests that, with further validation CTJ could be a promising herbal resource for prevention of atherosclerosis.

PANoptosis is a prominent cell death feature in thoracic aortic aneurysm or dissection

Thoracic aortic aneurysm and dissection (TAAD) is a devastating macrovascular disease, and its pathogenic mechanisms have not been well clarified. This study aimed to investigate the role of PANoptosis, which is newly defined programmed cell death (PCD) and characterized by pyroptosis, apoptosis, and necroptosis, in the pathogenesis of TAAD. We found that the expression of initiator factor Z-DNA binding protein 1 (ZBP1) and PANoptosis-related genes were upregulated in the β-aminopropionitrile (BAPN) + Angiotensin II (Ang II)-induced TAAD mice. Ang II stimuli enhanced the expression of ZBP1, promoted the generation of bioactive GSDMD (Gasdermin D) fragments, the cleavage of Caspase 3, and increased the phosphorylation of mixed lineage kinase domain-like pseudokinase (MLKL) in human aortic vascular smooth muscle cells (HASMCs), indicating the activation of hallmarks for PANoptosis. Moreover, ZBP1-mediated PANoptosis occurs in the aortic tissues of TAAD patients. These results highlight the significant role of PANoptosis in TAAD pathogenesis, suggesting ZBP1 and other PANoptosis-related genes as potential therapeutic targets for this condition.

Abstract P228: Kdm6a Regulates Extracellular Matrix Genes During Arterial Stiffening and High Blood Pressure in Female Mice

Arterial stiffness measured by pulse wave velocity (PWV) increases steeply during menopause due to estradiol decline. Longitudinal studies show higher PWV in postmenopausal women compared to age-matched men. Previously, we have demonstrated increased PWV in ovariectomized (OVX) and middle-aged female mice. However, the impact of sex chromosomes on PWV is unknown in female mice. We hypothesize that a decline in estradiol unleashes the female sex chromosomes effect that promotes arterial stiffening. This study used four core genotype mice (n=6-10/group) of XX or XY sex chromosomes left intact or OVX for 8 weeks. Arterial stiffening was assessed with pulse wave Doppler and 24-hour blood pressure by radiotelemetry. Carotid artery passive myography and vascular reactivity of 2 nd order mesenteric artery were assessed by pressure myography. To induce hypertension, XX and XY mice were treated with deoxycorticosterone acetate (DOCA; 50 mg/pellet) for 21 days and 1% saline. In vitro, isolated aortic vascular smooth muscle cells (VSMC) were stretched on collagen matrix or treated with Kdm6a inhibitor GSKJ-1. PWV was significantly increased in OVX than in gonadal intact XX and XY mice (P<0.001). However, an interaction effect was indicated with higher PWV in XX than XY OVX mice (4.9±0.1 vs. 4.2±0.2 m/s; P<0.001), indicating a sex chromosome effect. Carotid artery stress-strain curves showed a leftward shift of XX than XY mice, suggesting increased structural stiffness. Masson’s trichrome staining of the aorta showed increased collagen in the adventitia of XX than XY (30% vs. 26%; P=0.005) mice. Higher systolic blood pressure (SBP) was indicated in XX than in XY mice (Baseline; 123±3 vs. 116±4 mmHg; P=0.02). Aortic RNAseq showed increased X-linked genes, including Kdm6a, in XX compared to XY mice. Stretched aortic VSMC showed increased Col1a1 and decreased Itga4 and Lama2 genes reversed by GSKJ-1. DOCA-induced hypertension blunted the PWV difference between XX and XY mice; however, SBP was higher in XX than in XY mice (Week 1: 135±13 vs.127±3 mmHg; P=0.03), but not weeks 2 and 3. We observed impaired acetylcholine-mediated relaxation in hypertensive XX than XY mice (47% vs. 24%; P=0.03). Our data suggested that OVX mice with XX sex chromosomes promote arterial stiffening and high blood pressure, and Kdm6a regulates extracellular matrix genes in XX female VSMC. DOCA-induced hypertension promotes endothelial dysfunction and blunts PWV in ovariectomized XX and XY mice.

Abstract 76: CASC15 lncRNA as a Mediator of Vascular Senescence in the Renin-Angiotensin System

The renin-angiotensin system (RAS) regulates vascular function and maintains blood pressure homeostasis. Angiotensin-II (Ang-II) and angiotensin-1-7 (Ang-1-7) have opposing effects on vascular health. Ang-II promotes vascular senescence and pathophysiological remodeling by inducing oxidative stress, inflammation, vascular hypertrophy, and endothelial dysfunction. In contrast, Ang-1-7, known for its vasodilatory, anti-inflammatory, and antioxidative properties, counteracts these detrimental effects, enhancing endothelial function, reducing oxidative stress, and inhibiting inflammation. Our laboratory has identified the long non-coding RNA CASC15 as a critical regulatory element within the RAS. CASC15 expression is significantly repressed by Ang-II in aortic vascular smooth muscle cells (SMCs), showing a 59.25% decrease compared to the vehicle control (n=10, p=0.000333). Loss of CASC15 induces cellular senescence, increasing senescent cells from a mean of 14.09% (control inhibitor, n=9, SEM=4.824) to 57.79% (CASC15 inhibitor, n=9, SEM=6.420, p<0.0001), and leads to vascular hypertrophy, with cell area increasing from a mean of 46.89% (control inhibitor, n=8, SEM=9.229) to 221.2% (CASC15 inhibitor, n=9, SEM=67.53, p=0.0300). We hypothesize that CASC15 repression by Ang-II (1 µM) contributes to vascular senescence and increased DNA damage, while Ang-1-7 (1 µM) preserves CASC15 expression and mitigates these effects. Pro-senescent stressors like hydrogen peroxide and doxorubicin also repress CASC15 expression in SMCs. Using RT-qPCR, we observed decreases in CASC15 expression by 29.56% (hydrogen peroxide vs. vehicle, n=4, p=0.0790) and 44.09% (doxorubicin vs. vehicle, n=3, p=0.0733). Ang-1-7 alone did not significantly change CASC15 expression, but combined with Ang-II, it showed potential recovery from 40% repression to 94% recovery. Further research is needed. Immunofluorescence against gamma-H2AX revealed increased DNA damage in SMCs lacking CASC15, with positive foci increasing from 26.27% (control inhibitor, n=4, SEM=1.459) to 63.71% (CASC15 inhibitor, n=4, SEM=1.806, p=0.0009). In conclusion, CASC15 is a crucial regulator within the RAS. Its repression by Ang-II promotes vascular senescence, hypertrophy, and DNA damage, effects countered by Ang-1-7. Targeting CASC15 could offer new strategies for mitigating vascular aging and improving vascular health, particularly in chronic kidney disease.

Quercetin Attenuates KLF4-Mediated Phenotypic Switch of VSMCs to Macrophage-like Cells in Atherosclerosis: A Critical Role for the JAK2/STAT3 Pathway.

The objective of this study was to elucidate the protective role of quercetin in atherosclerosis by examining its effect on the phenotypic switch of vascular smooth muscle cells (VSMCs) to macrophage-like cells and the underlying regulatory pathways. Aorta tissues from apolipoprotein E-deficient (ApoE KO) mice fed a high-fat diet (HFD), treated with or without 100 mg/kg/day quercetin, were analyzed for histopathological changes and molecular mechanisms. Quercetin was found to decrease the size of atherosclerotic lesions and mitigate lipid accumulation induced by HFD. Fluorescence co-localization analysis revealed a higher presence of macrophage-like vascular smooth muscle cells (VSMCs) co-localizing with phospho-Janus kinase 2 (p-JAK2), phospho-signal transducer and activator of transcription 3 (p-STAT3), and Krüppel-like factor 4 (KLF4) in regions of foam cell aggregation within aortic plaques. However, this co-localization was reduced following treatment with quercetin. Quercetin treatment effectively inhibited the KLF4-mediated phenotypic switch in oxidized low-density lipoprotein (ox-LDL)-loaded mouse aortic vascular smooth muscle cells (MOVAS), as indicated by decreased expressions of KLF4, LGALS3, CD68, and F4/80, increased expression of alpha smooth muscle actin (α-SMA), reduced intracellular fluorescence Dil-ox-LDL uptake, and decreased lipid accumulation. In contrast, APTO-253, a KLF4 activator, was found to reverse the effects of quercetin. Furthermore, AG490, a JAK2 inhibitor, effectively counteracted the ox-LDL-induced JAK2/STAT3 pathway-dependent switch to a macrophage-like phenotype and lipid accumulation in MOVAS cells. These effects were significantly mitigated by quercetin but exacerbated by coumermycin A1, a JAK2 activator. Our research illustrates that quercetin inhibits the KLF4-mediated phenotypic switch of VSMCs to macrophage-like cells and reduces atherosclerosis by suppressing the JAK2/STAT3 pathway.

Ligustrazine alleviates the progression of coronary artery calcification by inhibiting caspase-3/GSDME mediated pyroptosis.

Coronary artery calcification (CAC) is an early marker for atherosclerosis and is mainly induced by the osteoblast-like phenotype conversion of vascular smooth muscle cells (VSMCs). Recent reports indicate that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis plays a significant role in the calcification of vascular smooth muscle cells (VSMCs), making it a promising target for treating calcific aortic valve disease (CAC). Ligustrazine, or tetramethylpyrazine (TMP), has been found effective in various cardiovascular and cerebrovascular diseases and is suggested to inhibit NLRP3-mediated pyroptosis. However, the function of TMP in CAC is unknown. Herein, influences of TMP on β-glycerophosphate (β-GP)-stimulated VSMCs and OPG-/- mice were explored. Mouse Aortic Vascular Smooth Muscle (MOVAS-1) cells were stimulated by β-GP with si- caspase-3, si- Gasdermin E (GSDME) or TMP. Increased calcification, reactive oxygen species (ROS) level, Interleukin-1beta (IL-1β) and Interleukin-18 (IL-18) levels, lactate dehydrogenase (LDH) release, enhanced apoptosis, and activated cysteine-aspartic acid protease-3 (caspase-3)/GSDME signaling were observed in β-GP-stimulated MOVAS-1 cells, which was sharply alleviated by si-caspase-3, si-GSDME or TMP. Furthermore, the impact of TMP on the β-GP-induced calcification and injury in MOVAS-1 cells was abolished by raptinal, an activator of caspase-3. Subsequently, OPG-/- mice were dosed with TMP or TMP combined with raptinal. Calcium deposition, increased nodules, elevated IL-1β and IL-18 levels, upregulated CASP3 and actin alpha 2, smooth muscle (ACTA2), and activated caspase-3/GSDME signaling in OPG-/- mice were markedly alleviated by TMP, which were notably reversed by the co-administration of raptinal. Collectively, TMP mitigated CAC by inhibiting caspase-3/GSDME mediated pyroptosis.

Characterization of Polyphenols in Baru (Dipteryx alata) and Their Effects on Palmitic Acid-Induced Oxidative Stress in Rat Aortic Vascular Smooth Muscle Cells

Characterization of Polyphenols in Baru (Dipteryx alata) and Their Effects on Palmitic Acid-Induced Oxidative Stress in Rat Aortic Vascular Smooth Muscle Cells

PCSK9 expression in fibrous cap possesses a marker for rupture in advanced plaque.

To date, PCSK9 inhibitors are well known for eliminating cardiac and cerebral artery ischemia events by lowering the serum lipid level. However, the pathophysiological value of in-plaque PCSK9 expression is still unclear. Advanced plaques removed by carotid endarterectomy were sectioned and stained to identify the PCSK9 expression pattern and its co-expression with rupture-relevant markers. To investigate the correlation of PCSK9 expression with regional blood shear flow, hemodynamic characteristics were analyzed using computational fluid dynamics, and representative parameters were compared between PCSK9 positive and negative staining plaques. To explore this phenomenon in vitro, human aortic vascular smooth muscle cells were used to overexpress and knock down PCSK9. The impacts of PCSK9 modulations on mechanical sensor activity were testified by western blot and immunofluorescence. Real-time polymerase chain reaction was used to evaluate the transcription levels of downstream rupture-prone effectors. PCSK9 distribution in plaque preferred cap and shoulder regions, residing predominantly in smooth muscle actin-positive cells. Cap PCSK9 expression correlated with fibrous cap thickness negatively and co-expressed with MMP-9, both pointing to the direction of plaque rupture. A hemodynamic profile indicated a rupture-prone feature of cap PCSK9 expression. In vitro, overexpression and knockdown of PCSK9 in human aortic vascular smooth muscle cells has positive modulation on mechanical sensor Yes-associated protein 1 (YAP) activity and transcription levels of its downstream rupture-prone effectors. Serial section staining verified in situ colocalization among PCSK9, YAP, and downstream effectors. Cap PCSK9 possesses a biomarker for rupture risk, and its modulation may lead to a novel biomechanical angle for plaque interventions.

High estrogen induces trans-differentiation of vascular smooth muscle cells to a macrophage-like phenotype resulting in aortic inflammation via inhibiting VHL/HIF1a/KLF4 axis.

Estrogen is thought to have a role in slowing down aging and protecting cardiovascular and cognitive function. However, high doses of estrogen are still positively associated with autoimmune diseases and tumors with systemic inflammation. First, we administered exogenous estrogen to female mice for three consecutive months and found that the aorta of mice on estrogen develops inflammatory manifestations similar to Takayasu arteritis (TAK). Then, in vitro estrogen intervention was performed on mouse aortic vascular smooth muscle cells (MOVAS cells). Stimulated by high concentrations of estradiol, MOVAS cells showed decreased expression of contractile phenotypic markers and increased expression of macrophage-like phenotypic markers. This shift was blocked by tamoxifen and Krüppel-like factor 4 (KLF4) inhibitors and enhanced by Von Hippel-Lindau (VHL)/hypoxia-inducible factor-1α (HIF-1α) interaction inhibitors. It suggests that estrogen-targeted regulation of the VHL/HIF-1α/KLF4 axis induces phenotypic transformation of vascular smooth muscle cells (VSMC). In addition, estrogen-regulated phenotypic conversion of VSMC to macrophages is a key mechanism of estrogen-induced vascular inflammation, which justifies the risk of clinical use of estrogen replacement therapy.

Significance of copper homeostasis in pathophysiology of human aortic vascular smooth muscle cells: Role in calcification and cell migration

Significance of copper homeostasis in pathophysiology of human aortic vascular smooth muscle cells: Role in calcification and cell migration

Fibronectin/α5 Integrin Contribute to Hypertension-Associated Arterial Ageing and Calcification through Affecting BMP2/MGP Imbalance and Enhancing Vascular Smooth Muscle Cell Phenotypic Transformation

Introduction: Hypertension can accelerate and aggravate the process of arterial ageing and calcification. However, the mechanism behind has yet to be well elucidated. Methods: Here, we monitored the dynamic changes of fibronectin (FN)/α5 integrin, bone morphogenetic protein 2/matrix Gla protein (BMP2/MGP), and Runx2 in the aorta of spontaneously hypertensive rats (SHRs) and thoracic aortic vascular smooth muscle cells (VSMCs), also the phenotypic transformation of VSMCs during the process of arterial ageing and calcification. Further, study on arterial ageing and calcification through antagonist experiments at the molecular level was explored. Results: We found extracellular FN and its α5 integrin receptor expressions were positively associated with arterial ageing and calcification in SHR during ageing, as well in VSMCs from SHR in vitro. Integrin receptor inhibitor of GRGDSP would delay this arterial ageing and calcification process. Moreover, the elevated FN and α5 integrin receptor expression evoked the disequilibrium of BMP2/MGP, where the expression of BMP2, a potent osteogenic inducer, increased while MGP, a calcification inhibitor, decreased. Furthermore, it was followed by the upregulation of Runx2 and the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Notably, BMP2 antagonist of rmNoggin was sufficient to ameliorate the ageing and calcification process of VSMCs and exogenous BMP2-adding accelerate and aggregate the process. Conclusion: Our study revealed that hypertension-associated arterial ageing and calcification might be a consequence that hypertension up-regulated FN and its high binding affinity integrin α5 receptor in the aortic wall, which in turn aggravated the imbalance of BMP2/MGP, promoted the transcription of Runx2, and induced the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Our study would provide insights into hypertension-associated arterial ageing and calcification and shed new light on the control of arterial calcification, especially for those with hypertension.

Nicotine Impairs Smooth Muscle cAMP Signaling and Vascular Reactivity.

This study aimed to determine nicotine's impact on receptor-mediated cyclic adenosine monophosphate (cAMP) synthesis in vascular smooth muscle (VSM). We hypothesize that nicotine impairs β adrenergic-mediated cAMP signaling in VSM, leading to altered vascular reactivity. The effects of nicotine on cAMP signaling and vascular function were systematically tested in aortic VSM cells and acutely isolated aortas from mice expressing the cAMP sensor TEpacVV (Camper), specifically in VSM (e.g., CamperSM). Isoproterenol (ISO)-induced β-adrenergic production of cAMP in VSM was significantly reduced in cells from second-hand smoke (SHS)-exposed mice and cultured wild-type VSM treated with nicotine. The decrease in cAMP synthesis caused by nicotine was verified in freshly isolated arteries from a mouse that had cAMP sensor expression in VSM (e.g., CamperSM mouse). Functionally, the changes in cAMP signaling in response to nicotine hindered ISO-induced vasodilation, but this was reversed by immediate PDE3 inhibition. These results imply that nicotine alters VSM β adrenergic-mediated cAMP signaling and vasodilation, which may contribute to the dysregulation of vascular reactivity and the development of vascular complications for nicotine-containing product users.

Abstract 3010: Inhibition Of Endothelial Cell-Mediated Efferocytosis Promotes Abdominal Aortic Aneurysm Formation

Introduction: Aortic wall inflammation and vascular smooth muscle (VSMC) apoptosis can lead to accumulation of apoptotic cells and debris during abdominal aortic aneurysm (AAA) formation. We hypothesize that dysregulation of MerTK receptor-mediated efferocytosis (clearance of apoptotic cell debris) by endothelial cells (ECs) can significantly contribute to aortic inflammation and vascular remodeling in AAA pathogenesis. Methods: Single-cell RNA sequencing of human AAA tissue from a published dataset (PMID: 33779682) was evaluated for cells of the aortic wall (VSMC/EC) identified via SingleR and cell-specific marker s. Differentially expressed genes (DEGs) were identified via edgeR using the QL F-test and efferocytosis-related genes (ERGs) were recognized using a list of 141 ERGs curated from GeneCards. MerTK expression was assessed in human aortic tissue and ECs via western blot and immunofluorescence. PKH67-labelled apoptotic VSMCs were overlayed on ECs to assess efferocytosis activity and visualized via confocal microscopy. In a murine elastase AAA model, endothelial-specific MerTK knockout ( Cdh5 Cre-ERT2 / MerTK fl/fl ) and littermate male mice were analyzed on postoperative day 14 for aortic diameter. Statistical analyses were performed by Welch’s T-test and p<0.05 was considered statistically significant. Results: ERGs (ANXA2, PHACTR1, PPARG, ADAM9, CD14, IL6) were differentially expressed (p<0.05, log 2 FC > |1|) between aortic wall cells of human AAAs and control. Human AAA tissue displayed a significant increase in MerTK protein compared to controls (1±0.1 vs. 1.51±0.4, n=6/group, p=0.01). The expression of MerTK in aortic ECs was confirmed by western blot and immunofluorescence(n=3). Pharmacologic inhibition of MerTK using UNC5293, a specific MerTK inhibitor, attenuated EC-mediated efferocytosis of apoptotic VSMCs (qualitative data; 10 images/group n=4). Importantly, EC-specific MerTK -/- mice had a significant increase in aortic diameter compared to littermate controls (155±40.4 vs. 103±30.9%, n=5-6/group, p=0.04) on day 14. Conclusion: EC-mediated efferocytosis can be regulated by MerTK to mediate AAA formation. Ongoing studies will delineate mechanisms of EC-MerTK signaling in AAA formation.

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.

Fenoldopam-mediated increase in autophagy associates with recruiting WIPI2 protein in early endosomes in vascular smooth muscle cells

Autophagy, a crucial cellular homeostatic process, has protective roles in the pathogenesis of hypertension. Dopamine, a monoamine neurotransmitter, regulates autophagy in the kidney and vascular system. However, the exact roles and mechanisms of dopaminergic system on the regulation of autophagy is not well known. In rat aortic vascular smooth muscle cells, fenoldopam (FEN, 25 nM/15 min), a D1-like receptor agonist, increased the ATG5 and LC3-II (a late autophagosome maker) protein expression. Consistently, FEN decreased p62/SQSTM1 protein expression. The FEN-mediated increase in ATG5 was attenuated by SCH39166 (1 μM/15 min), a D1- like receptor antagonist, indicating that FEN increased autophagy in the autophagosome formation stage through its D1-like receptors. FEN increased the protein expression of WD Repeat Domain, Phosphoinositide Interacting 2 (WIPI2) in a concentration- and time-dependent manner, which is important in the early steps of autophagosome formation. FEN (25 nM, 15 min) increased the WIPI2 puncta fluorescence intensity and the colocalization of WIPI2 with EEA1 (Veh: 7.12±1.1% vs FEN: 9.96±0.9%, N=5, P < 0.001, Student’s t test), a typical phosphatidylinositol-3-phosphate binding protein in early endosomes, indicating that WIPI2 recruitment in early endosomes is important in the early stage of FEN-mediated autophagic process. Taken together, FEN increases autophagy with WIPI2 recruitment in the early endosomes in vascular smooth muscle cells. This work was supported by US National Institutes of Health grants R01DK134574 and R01DK119652. 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.

Abstract 3066: Pro-Atherogenic Macrophages Induce The Upregulation Of MLKL And Cell Death In Vascular Smooth Muscle Cells During Atherogenic Stimulation

Background: A hallmark of advanced, rupture-prone atherosclerotic plaques is the presence of a necrotic core - a dense mass of pro-inflammatory and cellular debris. Traditionally, it has been thought that most of the foam cells within this core are derived from macrophages. However, recent advances in single cell technologies have revealed that vascular smooth muscle cells have the capacity to transdifferentiate into "macrophage-like foam cells", accounting for almost half of the cell content in advanced plaques. Our lab and others have discovered that a primary cause of the necrotic core is necroptotic cell death, where the mixed lineage kinase domain like pseudokinase MLKL is a key executioner of this process. While the contribution of necroptotic macrophage-derived foam cells to atherosclerotic lesion growth and instability has been fairly well-studied, the role of necroptotic vascular smooth muscle cell-derived foam cells in this process remains unclear. Hypothesis: MLKL regulates cell death differently in macrophages and vascular smooth muscle cells when challenged with pro-atherogenic stimuli. Methods and Results: Bone marrow-derived macrophages (BMDMs) and aortic vascular smooth muscle cells (VSMCs) were isolated from wildtype mice and treated with pro-atherogenic stimuli in the presence or absence of apoptotic and necroptotic inhibitors, zVAD and Necrostatin-1 (Nec1). BMDMs underwent cell death in response to treatment with oxLDL or LPS plus zVAD within 24 hours (as measured by LDH release and SYTOX assays) while VSMCs survive up to 72 hours. However, when VSMCs are treated with BMDM conditioned media in addition to pro-atherogenic stimuli, VSMCs begin to show higher relative rates of cell death compared to controls. Confocal microscopy and Western blots revealed that VSMCs treated with cell death ligands in conjunction with BMDM conditioned media showed an increase in relative expression of total and pMLKL- a hallmark of necroptosis, compared to VSMCs without conditioned media. Conclusions: Overall, this work suggests VSMCs are inherently resistant to necroptosis unless treated with conditioned media from BMDMs, which may suggest that macrophages have the capacity to prime necroptosis in VSMCs, likely via secreted factors.

O-GlcNAcylation regulates angiotensin II-dependent induction of thrombospondin 1

O-Linked attachment of β-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins is a highly dynamic and ubiquitous post-translational modification that plays an important role in altering the function, activity, subcellular localization, and stability of target proteins. It has been proposed that physiologically, O-GlcNAcylation serves primarily to modulate cellular signaling and transcription regulatory pathways in response to nutrients and stress. However, in the vascular physiology literature, O-GlcNAc has predominantly been shown to be detrimental. Our preliminary data has shown that O-GlcNAcylation can cause aortic stiffness, however, the mechanisms underlying this phenotype remain to be elucidated. The predominant mechanisms by which O-GlcNAcylation contributes to alterations in proteostasis include competition with other post-translational modifications as well as regulation of gene expression. Thrombospondin-1 (TSP1) is a multifunctional matrix glycoprotein that impacts extracellular function (ECM) and cell adhesion, migration, proliferation, and cell-cell interaction. Therefore, we hypothesized that TSP1 could be an important downstream mediator of O-GlcNAcylation in aortic vascular smooth muscle cells (VSMCs). To test this hypothesis, primary VSMCs from the thoracic aorta were isolated from 12-week-old female Wistar rats and submitted to angiotensin (ANG) II treatment (100 nM, 3h). Some cells were also treated in the presence of ANG II type 1 (AT1)-receptor antagonist losartan (10−5 M, 30 min). ANG II was chosen because it is a physiological stressor known to cause arterial stiffening. O-GlcNAcylation (RL2 antibody), O-GlcNAcase (OGA), and TSP1 protein amounts were analyzed by Western blot, and the results were expressed in arbitrary units (AU) and mean ±standard error mean. Unpaired Student's test T was used and p<0.05 was considered statistically significant. Genetic and pharmacological inhibition of O-GlcNAc was achieved through OGT silencing and inhibition with a shRNA (FunGene6) and OSMI-4 (10−5 M, 30 min), respectively. As hypothesized, ANG II significantly increased O-GlcNAc in aortic VSMC (0.3±0.03 vs 0.5±0.03 AU n=8, p<0.05) which was inhibited by losartan (0.1±0.02). Moreover, ANG II significantly decreased OGA expression (0.16±0.04 vs 1.16±0.19 AU n=4, p<0.05), the enzyme responsible for removing O-GlcNAc motifs, supporting the observed increase in O-GlcNAc expression. Importantly, ANG II also increased TSP1 protein expression (0.2±0.04 vs 2.0±0.4 n=4, p<0.05), however, shRNA (0.7±0.05) for OGT and OSMI-4 (0.06±0.08) prevented this increase. Overall, these data indicate that Ang II-AT1R signaling can cause O-GlcNAcylation and that TSP1 could be an important downstream mediator of O-GlcNAc-mediated aortic stiffness. National Institutes of Health (R00HL151889, P20GM109091-Pilot Project, and P20GM103641-Pilot Project). University of South Carolina Offce of Research ASPIRE Award (180950-23-64136). American Heart Association Postdoctoral Fellow (916031). 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.

Exploring the Impact of Nitric Oxide and Epidermal Growth Factor Receptor on Vascular Remodeling Induced by Mechanical Stretch

Hypertension is a key risk factor in many cardiovascular diseases like heart failure, kidney failure and vascular hypertrophy. Mechanical stretch/hypertension, promotes vascular smooth muscle cells remodeling by inducing leptin expression and other hypertrophic proteins like ERK1/2, AKT, and cofilin. Also the Epidermal growth factor receptor (EGFR) transactivation has shown to mediate vascular remodeling by enhancing VSMC hypertrophy, migration, and proliferation. Nitric oxide (NO) has been shown to be a critical factor for many cellular physiological function, it has the ability to induce VSMC relaxation leading to reduce hypertension. Moreover NO has received extensive attention as an anti- hypertrophic agent. Several Studies have shown that nitric oxide does not only promote vasodilation but it also inhibits VSMC proliferation. The major aim of this study was to investigate the inhibitory effect of NO and the effect of blocking the EGFR in mechanical stretch induced vascular remodeling. Methods: Rat portal veins (RPVs) were isolated and pretreated with/without NO donor (SNAP) or EGFR kinase blocker (AG1478) and then stretched to study their effects on mechanical stretch induced leptin synthesis and VSMC remolding. Western blot analysis was done to detect protein expression for leptin, adiponectin, eNOS p-ERK1/2 and p-AKT. Immunocytochemistry was performed on rat aortic vascular smooth muscle (RASMC) to detect GATA-4 nuclear translocation. Moreover, we used laser confocal microscopy on frozen RPV sections to detect the level of ROS formation and leptin. Finally, we performed qPCR for adiponectin, adipoR1 and adipoR2 mRNA. Results: In this study we showed the effect of mechanical stretch at different time points (1hour and 24 hours). Mechanical stretch-induced leptin expression was mediated by ERK1/2, AKT activations and ROS formation. These effects were associated with eNOS and adiponectin downregulation. Mechanical stretch had no effect on the expression of adiponectin receptors (adipoR1 and adipoR2). RASMCs pretreated with 3.1 nmol/L for 3 hours had induced GATA-4 nuclear translocation. Pre-incubation the RPV with SNAP significantly decreased mechanical stretch induced leptin expression, ERK1/2 and AKT phosphorylation. Moreover, SNAP significantly inhibited the adiponectin downregulation by mechanical stretch. Leptin-induced GATA-4 nuclear translocation was inhibited by SNAP. The involvement of EGFR transactivation in mechanical stretch-induced leptin expression was investigated using AG1478. Pretreatment of RPVs with AG1478 had no effect on mechanical stretch-induced early leptin expression (1 hour) while it inhibited mechanical stretch-induced late leptin expression (24 hours), ROS formation, and adiponectin and eNOS downregulation. However, treatment with AG1478 showed no effect on leptin-induced GATA-4 nuclear translocation. Conclusion: This study showed that NO has a direct inhibitory effect on mechanical stretch-induced AKT and ERK1/2 pathways and inhibits ROS formation that leads to vascular remodeling. On the other hand, EGFR transactivation was able to mediate mechanical stretch-induced vascular hypertrophy via ROS formation and leptin production, thus making EGFR and NO a promising therapeutic target for hypertension induced hypertrophy. This work was funded by Qatar University grants award UREP27-050-3-020 from Qatar National Research Fund (a member of Qatar Foundation) to AZ. 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.