Wild-type Aortas Research Articles

Abstract 31: Deacetylation Mimetic Mutation of Mitochondrial Superoxide Dismutase Attenuates Angiotensin II-Induced Hypertension by Protecting Against Oxidative Stress

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite the use of multiple drugs, likely due to mechanisms that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. Hypertension is associated with inactivation of key intrinsic mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to hyperacetylation but the role of specific SOD2 lysine residues has not been defined. Hypertension is associated with SOD2 acetylation at Lysine 68 (Circ Res. 2020;126(4):439-452) and we suggested that deacetylation mimetic mutation of K68 to Arginine in SOD2 inhibits vascular oxidative stress and attenuates hypertension. To test this hypothesis, we have developed a new deacetylation mimic SOD2-K68R mice. We performed in vivo studies in SOD2-K68R mice using angiotensin II (AngII) model of vascular dysfunction and hypertension. AngII infusion in wild-type mice induced vascular inflammation and oxidative stress, and increased blood pressure to 160 mm Hg. SOD2-K68R mutation completely prevented increase in mitochondrial superoxide, abrogated vascular oxidative stress, preserved endothelial nitric oxide production, protected vasorelaxation, and attenuated AngII-induced hypertension. AngII and cytokines contribute to vascular oxidative stress and hypertension. Treatment of wild-type aortas with AngII and cytokines in organoid culture increased mitochondrial superoxide by 2-fold which was completely prevented in aortas isolated from SOD2-K68R mice. Interestingly, ex vivo treatment of aortas isolated from hypertensive mice with Sirtuin activator resveratrol reduces SOD2 acetylation, inhibits mitochondrial superoxide, and rescues endothelial nitric oxide. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and pathogenesis of hypertension. We conclude that strategies to reduce SOD2 acetylation may have therapeutic potential in the treatment of vascular dysfunction and hypertension.

AT1b receptors contribute to regional disparities in angiotensin II mediated aortic remodelling in mice.

The renin-angiotensin system plays a key role in regulating blood pressure, which has motivated many investigations of associated mouse models of hypertensive arterial remodelling. Such studies typically focus on histological and cell biological changes, not wall mechanics. This study explores tissue-level ramifications of chronic angiotensin II infusion in wild-type (WT) and type 1b angiotensin II (AngII) receptor null (Agtr1b -/-) mice. Biaxial biomechanical and immunohistological changes were quantified and compared in the thoracic and abdominal aorta in these mice following 14 and 28 days of angiotensin II infusion. Preliminary results showed that changes were largely independent of sex. Associated thickening and stiffening of the aortic wall in male mice differed significantly between thoracic and abdominal regions and between genotypes. Notwithstanding multiple biomechanical changes in both WT and Agtr1b -/- mice, AngII infusion caused distinctive wall thickening and inflammation in the descending thoracic aorta of WT, but not Agtr1b -/-, mice. Our study underscores the importance of exploring differential roles of receptor-dependent angiotensin II signalling along the aorta and its influence on distinct cell types involved in regional histomechanical remodelling. Disrupting the AT1b receptor primarily affected inflammatory cell responses and smooth muscle contractility, suggesting potential therapeutic targets.

Deacetylation mimetic mutation of mitochondrial SOD2 attenuates ANG II-induced hypertension by protecting against oxidative stress and inflammation.

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite the use of multiple drugs, likely because of mechanisms that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. Hypertension is associated with inactivation of key intrinsic mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to hyperacetylation, but the role of specific SOD2 lysine residues has not been defined. Hypertension is associated with SOD2 acetylation at lysine 68, and we suggested that deacetylation mimetic mutation of K68 to arginine in SOD2 inhibits vascular oxidative stress and attenuates hypertension. To test this hypothesis, we have developed a new deacetylation mimetic SOD2-K68R mice. We performed in vivo studies in SOD2-K68R mice using angiotensin II (ANG II) model of vascular dysfunction and hypertension. ANG II infusion in wild-type mice induced vascular inflammation and oxidative stress and increased blood pressure to 160 mmHg. SOD2-K68R mutation completely prevented increase in mitochondrial superoxide, abrogated vascular oxidative stress, preserved endothelial nitric oxide production, protected vasorelaxation, and attenuated ANG II-induced hypertension. ANG II and cytokines contribute to vascular oxidative stress and hypertension. Treatment of wild-type aortas with ANG II and cytokines in organoid culture increased mitochondrial superoxide twofold, which was completely prevented in aortas isolated from SOD2-K68R mice. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and pathogenesis of hypertension. We conclude that strategies to reduce SOD2 acetylation may have therapeutic potential in the treatment of vascular dysfunction and hypertension.NEW & NOTEWORTHY Essential hypertension is associated with hyperacetylation of key mitochondrial antioxidant SOD2; however, the pathophysiological role of SOD2 acetylation has not been defined. Our animal study of angiotensin II hypertension model shows that deacetylation mimetic SOD2-K68R mutation prevents pathogenic increase in vascular mitochondrial superoxide, abrogates vascular oxidative stress, preserves endothelial nitric oxide, protects endothelial-dependent vasorelaxation, and attenuates hypertension. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and the pathogenesis of hypertension.

Type 1 Diabetes Impairs Endothelium-Dependent Relaxation Via Increasing Endothelial Cell Glycolysis Through Advanced Glycation End Products, PFKFB3, and Nox1-Mediated Mechanisms.

Type 1 diabetes (T1D) is a major cause of endothelial dysfunction. Although cellular bioenergetics has been identified as a new regulator of vascular function, whether glycolysis, the primary bioenergetic pathway in endothelial cells (EC), regulates vascular tone and contributes to impaired endothelium-dependent relaxation (EDR) in T1D remains unknown. Experiments were conducted in Akita mice with intact or selective deficiency in EC PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), the main regulator of glycolysis. Seahorse analyzer and myography were employed to measure glycolysis and mitochondrial respiration, and EDR, respectively, in aortic explants. EC PFKFB3 (Ad-PFKFB3) and glycolysis (Ad-GlycoHi) were increased in situ via adenoviral transduction. T1D increased EC glycolysis and elevated EC expression of PFKFB3 and NADPH oxidase Nox1 (NADPH oxidase homolog 1). Functionally, pharmacological and genetic inhibition of PFKFB3 restored EDR in T1D, while in situ aorta EC transduction with Ad-PFKFB3 or Ad-GlycoHi reproduced the impaired EDR associated with T1D. Nox1 inhibition restored EDR in aortic rings from Akita mice, as well as in Ad-PFKFB3-transduced aorta EC and lactate-treated wild-type aortas. T1D increased the expression of the advanced glycation end product precursor methylglyoxal in the aortas. Exposure of the aortas to methylglyoxal impaired EDR, which was prevented by PFKFB3 inhibition. T1D and exposure to methylglyoxal increased EC expression of HIF1α (hypoxia-inducible factor 1α), whose inhibition blunted methylglyoxal-mediated EC PFKFB3 upregulation. EC bioenergetics, namely glycolysis, is a new regulator of vasomotion and excess glycolysis, a novel mechanism of endothelial dysfunction in T1D. We introduce excess methylglyoxal, HIF1α, and PFKFB3 as major effectors in T1D-mediated increased EC glycolysis.

Abstract 282: Upregulation Of Receptor Interacting Protein Kinase-3 Augments Abdominal Aortic Aneurysms

Objective: Aortic smooth muscle cell (SMC) depletion is a major pathological feature of abdominal aortic aneurysm (AAA). Our lab has previously reported elevated levels of Receptor Interacting Protein Kinase-3 (RIPK3), a major mediator of necroptosis, in human AAA tissues. Pharmacologic inhibition of RIPK3 or deletion of the Ripk3 gene prevents aortic SMC death and attenuates aneurysm growth in murine models. In the current study, we tested the hypothesis that manipulation of Ripk3 gene expression would alter AAA development. Methods: We manipulated Ripk3 gene expression by mutating a conserved putative transcription enhancer site using CRISPR-Cas9-mediated gene editing. AAA was induced in 8-week old male CRISPR mutated (CM) and wild-type (WT) mice by perivascular application of CaCl 2 or NaCl (sham). Aortic tissues were harvested on days 4 or 28 post-surgery and analyzed by immunohistochemistry (IHC). Primary aortic SMCs isolated from both groups were stimulated with inflammation-related cytokines and Ripk3 mRNA was examined by RT-PCR. Results: Both CM and WT mice developed AAA in response to CaCl 2 . Change in aortic diameter was greater in CM mice than WT at 28 days (66.81±4.512 vs 44.87±6.685%, P =0.0108). IHC showed CM aneurysm tissue to exhibit fewer SMCs and a greater number of non-SMC cells in the tunica media, presumably infiltrated immune cells. Furthermore, mutant tissues contained higher levels of RIPK3 positivity than WT aortas. Consistently, CM aortic SMCs expressed more Ripk3 mRNA than WT SMCs, both with vehicle and following stimulation with IL-1β, IL-25, and IL-33. Conclusions: Our data show a positive correlation between Ripk3 expression and aneurysm size. We are currently analyzing data from experiments aimed to prove the causal relationship between Ripk3 gene editing, SMC necroptosis, inflammation, and aneurysm growth. This study further illustrates the potential of RIPK3 as a therapeutic target of AAA.

Abstract 281: A20 (tnfaip3) Knockdown Protects From Abdominal Aortic Aneurysms By Limiting Extra-cellular Matrix Degradation And Maintaining Aortic Smooth Muscle Cell Mass

Background: Our lab has recently demonstrated that haploinsufficiency of A20/TNFAIP3, a potent anti-inflammatory/NFκB inhibitory gene, protects mice from abdominal aortic aneurysms (AAA) by enhancing TGFβ signaling and modulating smooth muscle cell (SMC) differentiation and apoptosis. Here, we explored the impact of A20 knockdown on TGFβ-mediated SMC proliferation and extra cellular matrix (ECM) degradation, both involved in AAA pathogenesis. Methods: AAA was induced by periadventitial elastase application to the infrarenal aorta of both A20 wild-type (WT) and A20 heterozygous (Het) 10-week-old mice who received 0.2% BAPN in their drinking water. Aortae were harvested 28 days after for immunohistochemistry (IHC) analysis of SMC proliferation and ECM markers. In vitro , human aortic SMC cells (HAoSMC) were transduced with rAd.ShRNA A20 (siA20), resulting in 50-70% A20 knockdown, or control rAd.ShRNA Scramble (siScr). HAoSMC were starved for 24 hours, then treated with TGFβ. Proliferation was analyzed by MTS assay, and cell extracts assayed by Western blot (WB) for cell-cycle markers. Results: A20 haplo-insufficiency protected from AAA formation by: 1) decreasing ECM degradation, as gauged by significantly lower MMP-9 expression (n=3, p<0.01), and 2) promoting vessel wall thickening by increasing SMC proliferation (ki67+ nuclei/vessel wall, n=4, p<0.01). Increased SMC proliferation in Het vs. WT aortae correlated with significantly lower expression of the cell-cycle brake, p21 (p21+ nuclei/vessel wall, n=3-7, p<0.05). Akin mouse aortae, A20 knockdown significantly increased TGFβ-mediated HAoSMC proliferation, assessed by the MTS assay (siA20 vs. siScr, n=5, p<0.05). This associated with significantly higher protein levels of the proliferation regulator phospho-ERK1/2 in siA20 vs. siScr-transduced HAoSMC (WB, n=4, p<0.001). Conclusion: Our findings qualify the protective effect of A20 knockdown against AAA development by preserving ECM integrity and promoting TGFβ-mediated SMC proliferation. These targets of A20 knockdown were validated in HAoSMC, further supporting our pursuit of A20-siRNA based therapies to prevent or limit progression of AAA.

A20 (TNFAIP3) Knockdown Protects from Abdominal Aortic Aneurysms by Promoting Smooth Muscle Cell Differentiation and Resistance to Apoptosis

Introduction: The implication of inflammation in the pathogenesis of abdominal aortic aneurysms (AAA) remains unresolved. Our laboratory has previously established the potent atheroprotective properties of the anti-inflammatory gene A20. In this study, we investigated A20’s implication in the development of AAA. Methods: Aneurysms were induced by periadventitial application of elastase to the infrarenal aorta of 10-week-old A20 wild-type (WT) and A20-deficient heterozygous (Het) C57BL/6 mice supplemented with 0.2% BAPN drinking water. Aortic diameter was measured by ultrasound before and 28 days (d) after. Aortae were harvested at 28d for morphology, immunohistochemistry (IHC) and molecular profiling (qPCR). Results: Het mice were protected from AAA formation. Percent increase in aortic diameter was significantly lower in Het vs WT mice (<50% vs >120%, n = 13-17, p < 0.01). At the molecular level, protection from AAA in Het vs WT aortae corresponded with significantly i) higher levels of TGFβ (qPCR, n = 17-18, p < 0.01), a promoter of SMC differentiation, and downstream SMAD3 phosphorylation (IHC, n = 5; p < 0.0001); and ii) reduced rate of SMC apoptosis (TUNEL, n = 4, p < 0.01). As anticipated, knockdown of A20, a potent inhibitor of NF-κB and IFNγ signaling, significantly increased mRNA levels of the inflammatory markers IκBα, p65, MCP1, IFNγ, and IP10 in Het vs WT (n = 17-18, p < 0.05). Conclusion: This is the first report that A20 knockdown protects from AAA by increasing TGFβ signaling and reducing SMC apoptosis, despite promoting inflammation. Pilot studies validate that A20 polymorphisms lowering its expression slow AAA progression, supporting our pursuit of A20-siRNA gene therapies.

DCBLD2 regulates vascular hyperplasia by modulating the platelet derived growth factor receptor-β endocytosis through Caveolin-1 in vascular smooth muscle cells.

DCBLD2 is a neuropilin-like transmembrane protein that is up-regulated during arterial remodeling in humans, rats, and mice. Activation of PDGFR-β via PDGF triggers receptor phosphorylation and endocytosis. Subsequent activation of downstream signals leads to the stimulation of phenotypic conversion of VSMCs and arterial wall proliferation, which are common pathological changes in vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. In this study, we hypothesized that DCBLD2 regulates neointimal hyperplasia through the regulation of PDGFR-β endocytosis of vascular smooth muscle cells (VSMCs) through Caveolin-1 (Cav-1). Compared with wild-type (WT) mice or control littermate mice, the germline or VSMC conditional deletion of the Dcbld2 gene resulted in a significant increase in the thickness of the tunica media in the carotid artery ligation. To elucidate the underlying molecular mechanisms, VSMCs were isolated from the aorta of WT or Dcbld2-/- mice and were stimulated with PDGF. Western blotting assays demonstrated that Dcbld2 deletion increased the PDGF signaling pathway. Biotin labeling test and membrane-cytosol separation test showed that after DCBLD2 was knocked down or knocked out, the level of PDGFR-β on the cell membrane was significantly reduced, while the amount of PDGFR-β in the cytoplasm increased. Co-immunoprecipitation experiments showed that after DCBLD2 gene knock-out, the binding of PDGFR-β and Cav-1 in the cytoplasm significantly increased. Double immunofluorescence staining showed that PDGFR-β accumulated Cav-1/lysosomes earlier than for control cells, which indicated that DCBLD2 gene knock-down or deletion accelerated the endocytosis of PDGF-induced PDGFR-β in VSMCs. In order to confirm that DCBLD2 affects the relationship between Cav-1 and PDGFR-β, proteins extracted from VSMCs cultured in vitro were derived from WT and Dcbld2-/- mice, whereas co-immunoprecipitation suggested that the combination of DCBLD2 and Cav-1 reduced the bond between Cav-1 and PDGFR-β, and DCBLD2 knock-out was able to enhance the interaction between Cav-1 and PDGFR-β. Therefore, the current results suggest that DCBLD2 may inhibit the caveolae-dependent endocytosis of PDGFR-β by anchoring the receptor on the cell membrane. Based on its ability to regulate the activity of PDGFR-β, DCBLD2 may be a novel therapeutic target for the treatment of cardiovascular diseases.

Role of SERCA3-dependent calcium signaling in mouse aortic function

Calcium homeostasis has a major role in vascular reactivity. The Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCAs) contribute to generate intracellular Ca2+-stores that are mobilized during functional responses. The SERCA2 family is described as the most important in vascular cells, but SERCA3 has been reported to be co-expressed, with SERCA3a mainly at the endothelial level and SERCA3b at the smooth muscle level. Our objective is to evaluate the role of the SERCA3-dependent calcium pathway in vascular reactivity. For experiments, aortic rings were collected from wild-type (WT) and SERCA3-deficient (SERCA3-KO) mice (2–3 month-old male) and mounted in an organ bath system. Contractile and relaxant agents were applied to compare the in vitro aortic reactivity in both animal groups. The contractile responses to a depolarizing high-potassium solution or to cumulative increasing concentrations of serotonin (a 5-HT agonist, 10-9 to 10-5 M) or U46619 (a TXA2 analog, 10-10 to 3.10-6 M) were similar in SERCA3-KO and WT aorta. By contrast the response to phenylephrine (an α1-adrenergic agonist, 10-9 to 3.10-5 M) was significantly decreased in SERCA3-KO compared to WT aorta (N = 8-8; P < 0.05, 2-way ANOVA). However, in aorta denuded from endothelium, the contractile response to phenylephrine was similar in SERCA3-KO and WT vessels. In phenylephrine-precontracted aorta, the relaxant responses to acetylcholine (a muscarinic agonist, 10-9 to 3.10-5 M) or to isoproterenol (a b-adrenergic agonist, 10-10 to 10-5 M) were similar in both groups. The concentration-response curve to DEA-NOate (a NO donor, 10-11 to 10-5 M) was shifted to the right in SERCA3-KO compared to WT aortas, with a slight increase in EC50 (N = 9-8; P < 0.05, t -test). When the endothelium was removed, the effect to DEA-NOate was similar in both groups. We observed that SERCA3-KO aorta exhibit a major decrease in the amplitude of the α1-adrenergic contractile response, and in the sensitivity to the relaxant NO donor. These alterations are abolished in the absence of a functional endothelium. These preliminary data suggest that there is an influence of SERCA3 protein on the NO signaling pathway in mouse aorta.

Sirt1 overexpression attenuates Western-style diet-induced aortic stiffening in mice.

Increased arterial stiffness is a cardiovascular disease risk factor in the setting of advancing age and Western diet (WD) induced obesity. Increases in large artery stiffness, as measured by pulse wave velocity (PWV), occur within 8 weeks of WD feeding in mice. Sirtuin‐1 (Sirt1), a NAD‐dependent deacetylase, regulates cellular metabolic activity and activation of this protein has been associated with vasoprotection in aged mice. The aim of the study was to elucidate the effect of global Sirt1 overexpression (Sirttg) on WD‐induced arterial stiffening. Sirt1 overexpression did not influence PWV in normal chow (NC) fed mice. However, PWV was higher in wild‐type (WT) mice (p < 0.04), but not in Sirttg mice, after 12 weeks of WD and this effect was independent of changes in blood pressure or the passive pressure diameter relation in the carotid artery. Overexpression of Sirt1 was associated with lower collagen and higher elastin mRNA expression in the aorta of WD fed mice (both p < 0.05). Although MMP2 and MMP3 mRNA were both upregulated in WT mice after WD (both p < 0.05), this effect was reversed in Sirttg mice compared to WT mice fed WD (both p < 0.05). Surprisingly, histologically assessed collagen and elastin quality were unchanged in the aortas of WT or Sirttg mice after WD. However, Sirttg mice were protected from WD‐induced glucose intolerance, although there was no difference in insulin tolerance between groups. These findings demonstrate a vasoprotective effect of Sirt1 overexpression that limits the increase in arterial stiffness in response to consumption of a WD.

MFG-E8 Reduces Aortic Intimal Proliferation in a Murine Model of Transplant Vasculopathy.

Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.

Sirt7 Deficiency Attenuates Neointimal Formation Following Vascular Injury by Modulating Vascular Smooth Muscle Cell Proliferation.

Sirt7 is a recently identified sirtuin and has important roles in various pathological conditions, including cancer progression and metabolic disorders. It has previously been reported that Sirt7 is a key molecule in acute myocardial wound healing and pressure overload-induced cardiac hypertrophy. In this study, the role of Sirt7 in neointimal formation after vascular injury is investigated. Systemic (Sirt7-/-) and smooth muscle cell-specific Sirt7-deficient mice were subjected to femoral artery wire injury. Primary vascular smooth muscle cells (VSMCs) were isolated from the aorta of wild type (WT) and Sirt7-/-mice and their capacity for cell proliferation and migration was compared. Sirt7 expression was increased in vascular tissue at the sites of injury. Sirt7-/-mice demonstrated significant reduction in neointimal formation compared to WT mice. In vitro, Sirt7 deficiency attenuated the proliferation of serum-induced VSMCs. Serum stimulation-induced upregulation of cyclins and cyclin-dependent-kinase 2 (CDK2) was significantly attenuated in VSMCs of Sirt7-/-compared with WT mice. These changes were accompanied by enhanced expression of the microRNA 290-295 cluster, the translational negative regulator of CDK2, in VSMCs of Sirt7-/-mice. It was confirmed that smooth muscle cell-specific Sirt7-deficient mice showed significant reduction in neointima compared with control mice. Sirt7 deficiency attenuates neointimal formation after vascular injury. Given the predominant role in vascular neointimal formation, Sirt7 is a potentially suitable target for treatment of vascular diseases.

Abstract 12444: Smooth Muscle-Specific Deletion of Perk Prevents Atherosclerotic Plaque Formation in Mice

Introduction: Lineage tracing and single cell RNA sequencing (scRNA-Seq) studies have identified a subset of phenotypically modulated vascular smooth muscle cells (SMCs) that appear with atherosclerotic plaque formation in hyperlipidemic mice. These modulated SMCs (mSMCs) are de-differentiated, and increase expression of macrophage, fibroblast and stem cell markers. We previously showed that SMCs exposed to free cholesterol in vitro activate an endoplasmic reticulum unfolded protein response (UPR) and assume an mSMC phenotype, and blocking Perk specifically abolishes this SMC modulation. Methods: Here, we sought to define the role of Perk in atherosclerotic plaque formation using SMC-specific Perk knockout in C57BL6 mice ( Perk SMC-KO ) and inducing hyperlipidemia by PSCK9 DY overexpression and a high fat diet for 12 weeks. Results: Aortic Oil Red O staining and en face aortic analysis revealed no plaque formation in the Perk SMC-KO aorta, with the exception of some small plaques in the root, whereas the wild-type (WT) aortas had extensive plaque, especially in the aortic arch (N = 10, p&lt;0.0001, Fig. 1A ). scRNA-Seq analysis of aortic tissue from the root to the arch from these mice showed no major differences in cell clusters between Perk SMC-KO and WT mice at baseline ( Fig. 1B ). In hyperlipidemic WT mice, SMC-derived cluster 8 expands and a new cluster 7 appears (circled in red, Fig. 1B ). In Perk SMC-KO mice, cluster 8 is similarly expanded with hyperlipidemia, while cluster 7 is completely absent ( Fig. 1B , bottom right). Cluster 7 in the WT atherosclerotic aortas is the only cluster with Perk expression and has significantly increased expression of macrophage markers and a subset of fibroblast-specific markers when compared to cluster 8. Conclusions: These data indicate that SMC phenotypic modulation plays a major role in atherosclerotic plaque formation and Perk is a key regulator of the SMC modulation that specifically contributes to atherosclerotic plaque burden.

Apelin expression deficiency in mice contributes to vascular stiffening by extracellular matrix remodeling of the aortic wall

Numerous recent studies have shown that in the continuum of cardiovascular diseases, the measurement of arterial stiffness has powerful predictive value in cardiovascular risk and mortality and that this value is independent of other conventional risk factors, such as age, cholesterol levels, diabetes, smoking, or average blood pressure. Vascular stiffening is often the main cause of arterial hypertension (AHT), which is common in the presence of obesity. However, the mechanisms leading to vascular stiffening, as well as preventive factors, remain unclear. The aim of the present study was to investigate the consequences of apelin deficiency on the vascular stiffening and wall remodeling of aorta in mice. This factor freed by visceral adipose tissue, is known for its homeostasic role in lipid and vascular metabolisms, or again in inflammation. We compared the level of metabolic markers, inflammation of white adipose tissue (WAT), and aortic wall remodeling from functional and structural approaches in apelin-deficient and wild-type (WT) mice. Apelin-deficient mice were generated by knockout of the apelin gene (APL-KO). From 8 mice by groups, aortic stiffness was analyzed by pulse wave velocity measurements and by characterizations of collagen and elastic fibers. Mann–Whitney statistical test determined the significant data (p < 5%) between groups. The APL-KO mice developed inflammation, which was associated with significant remodeling of visceral WAT, such as neutrophil elastase and cathepsin S expressions. In vitro, cathepsin S activity was detected in conditioned medium prepared from adipose tissue of the APL-KO mice, and cathepsin S activity induced high fragmentations of elastic fiber of wild-type aorta, suggesting that the WAT secretome could play a major role in vascular stiffening. In vivo, remodeling of the extracellular matrix (ECM), such as collagen accumulation and elastolysis, was observed in the aortic walls of the APL-KO mice, with the latter associated with high cathepsin S activity. In addition, pulse wave velocity (PWV) and AHT were increased in the APL-KO mice. The latter could explain aortic wall remodeling in the APL-KO mice. The absence of apelin expression, particularly in WAT, modified the adipocyte secretome and facilitated remodeling of the ECM of the aortic wall. Thus, elastolysis of elastic fibers and collagen accumulation contributed to vascular stiffening and AHT. Therefore, apelin expression could be a major element to preserve vascular homeostasis.

Abstract MP01: Deacetylation Of Mitochondrial Cyclophilin D K166 Inhibits Cytokine-induced Oxidative Stress And Attenuates Hypertension

We have previously reported that depletion Cyclophilin D (CypD), a regulatory subunit of mitochondrial permeability transition pore, improves vascular function and attenuates hypertension, however, specific regulation of CypD in hypertension is not clear. Analysis of human arterioles from hypertensive patients did not reveal alterations in CypD levels but showed 3-fold increase in CypD acetylation. We hypothesized that CypD-K166 acetylation promotes vascular oxidative stress and hypertension, and measures to reduce CypD acetylation can improve vascular function and reduce hypertension. Essential hypertension and animal models of hypertension are linked to inactivation of mitochondrial deacetylase Sirt3 by highly reactive lipid oxidation products, isolevuglandins (isoLGs), and supplementation of mice with mitochondria targeted scavenger of isoLGs, mito2HOBA, improves CypD deacetylation. To test the specific role of CypD-K166 acetylation, we developed CypD-K166R deacetylation mimic mutant mice. Mitochondrial respiration, vascular function and systolic blood pressure in CypD-K166R mice was similar to wild-type C57Bl/6J mice. Meanwhile, angiotensin II-induced hypertension was substantially attenuated in CypD-K166R mice (144 mmHg) compared with wild-type mice (161 mmHg). Angiotensin II infusion in wild-type mice significantly increased mitochondrial superoxide, impaired endothelial dependent relaxation, and reduced the level of endothelial nitric oxide which was prevented in angiotensin II-infused CypD-K166R mice. Hypertension is linked to increased levels of inflammatory cytokines TNFα and IL-17A promoting vascular oxidative stress and end-organ damage. We have tested if CypD-K166R mice are protected from cytokine-induced oxidative stress. Indeed, ex vivo incubation of aorta with the mixture of angiotensin II, TNFα and IL-17A (24 hours) increased mitochondrial superoxide by 2-fold in wild-type aortas which was abrogated in CypD-K166R mice. These data support the pathophysiological role of CypD acetylation in inflammation, oxidative stress and hypertensive end-organ damage. We propose that targeting CypD acetylation may have therapeutic potential in treatment of vascular dysfunction and hypertension.

DRD4 (Dopamine D4 Receptor) Mitigate Abdominal Aortic Aneurysm via Decreasing P38 MAPK (mitogen-activated protein kinase)/NOX4 (NADPH Oxidase 4) Axis-Associated Oxidative Stress.

[Figure: see text].

Role of the PKA regulatory subunit RIα in the contractile tone of the aorta

Protein kinase A (PKA) is a key enzyme activated by binding of cAMP. PKA induces smooth muscle relaxation and vasodilatation. The PKA-RIα knock-in heterozygous mice, [R368X]/[+] (KI), leading to the generation of a truncated RIα and the repression of PKA activation (Le Stunff et al., 2017, J Bone Miner Res ) may help decipher the contribution of PKA-RIα in vascular system which has remained elusive. Our aim is to document the expression of PKA-RIα in mouse aorta and to decipher its role in arterial tone by using wild-type (WT) and KI mice. Aortas were harvested from adult male WT and KI mice. PKA subunits expressions were studied by immunoblot. Vascular reactivity was tested by mounting the aorta on a wire-myograph. The internal circumference (IC) was recorded. Concentration-contractile responses were performed for K + (20–120 mM), U46619 (1 nM–3 μM) and phenylephrine (PE, 1 nM–3 μM). Vasorelaxant responses to acetylcholine (ACh, 1 nM–10 μM) and to L858051 (adenylate cyclase activator, 10 nM–30 μM) were studied in aorta contracted with PE. All groups included 5–6 mice. In WT aorta, the anti-PKA-RIα antibody yielded a signal at 49 kDa, as expected. In KI mice, mutant allele expression was confirmed by an additional signal at 47 kDa. RIIα and Cα expressions were similar in both strains. IC was 20% smaller in KI mice than in WT. The contractile response to K + and PE were similar in both groups, whereas the response to U46619 was stronger in mutant aorta with a higher E max . ACh and L858051 relaxed aortas similarly in both groups. These preliminary data show that PKA-RIα is expressed in mouse aorta. The smaller IC of aorta in [R368X]/[+] mice is consistent with their smaller size (Le Stunff et al.). The stronger constriction to U46619 in mutant mice may be explained by reduced PKA activity compared to WT. The similar relaxation to L858051 in mutant mice maybe due to redundancy of PKA-RIα in mouse aorta.

Abstract 15061: Essential Hypertension is Linked to Acetylation of Mitochondrial Superoxide Dismutase and Deacetylation Mimetic Mutation of Lysine 68 to Arginine Protects From Oxidative Stress and Hypertension

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs, likely due to mechanisms contributing to blood pressure elevation that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. We found that hypertension is associated with inactivation of key mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to acetylation of lysine residues at the catalytic center. The role of specific SOD2 lysine residues in hypertension, however, has not been defined. Hypothesis: We proposed that inactivation of key intrinsic antioxidant, SOD2, in hypertension is linked to acetylation of Lysine 68, and mutation of K68 to Arginine mimics SOD2 deacetylation, inhibits vascular oxidative stress and attenuates angiotensin II-induced hypertension. To test this hypothesis, we have investigated SOD2 acetylation in arterioles from patients with essential hypertension and developed a new deacetylation mimic SOD2 mutant K68R mice (SOD2-K68R). Western blot of arterioles isolated from human mediastinal fat showed 3-fold increase in SOD2 acetylation in hypertensive patients compared with normotensive subjects while SOD2 levels were not affected. To define the functional significance of K68 acetylation we performed studies in vivo in SOD2-K68R mice using angiotensin II model of vascular dysfunction and hypertension. Angiotensin II infusion in wild-type C57Bl/6J mice induced vascular inflammation and oxidative stress, and increased blood pressure to 160 mm Hg. Mutation of Lysine 68 to Arginine in SOD2-K68R mice completely prevented the increase in mitochondrial superoxide and significantly attenuated the angiotensin II induced hypertension (135 mm Hg). Angiotensin II and TNFα co-operatively induce SOD2 acetylation and hypertension. Treatment of wild-type aortas with angiotensin II and TNFα in organoid culture increased mitochondrial superoxide by 2-fold which was completely prevented in aortas isolated from SOD2-K68R mice. Conclusions: These data support an important role of SOD2-K68 acetylation in hypertension and targeting Sirt3-mediated deacetylation of SOD2 may have therapeutic potential.

High-cholesterol diet during pregnancy induces maternal vascular dysfunction in mice: potential role for oxidized LDL-induced LOX-1 and AT1 receptor activation.

The lectin-like oxidized low-density-lipoprotein (oxLDL) receptor-1 (LOX-1) has been shown to induce angiotensin II (AngII) type 1 receptor (AT1) activation, contributing to vascular dysfunction. Preeclampsia is a pregnancy complication characterized by vascular dysfunction and increased LOX-1 and AT1 activation; however, whether LOX-1 and AT1 activity contributes to vascular dysfunction in preeclampsia is unknown. We hypothesized that increased oxLDL levels during pregnancy lead to LOX-1 activation and subsequent AT1 activation, resulting in vascular dysfunction. Pregnant wild-type (WT) and transgenic LOX-1 overexpressing (LOX-1tg) mice were fed a control diet (CD) or high-cholesterol diet (HCD, to impair vascular function) between gestational day (GD) 13.5-GD18.5. On GD18.5, AngII-induced vasoconstriction and methylcholine (MCh)-induced endothelium-dependent vasodilation responses were assessed in aortas and uterine arteries. HCD decreased fetal weight and increased circulating oxLDL/cholesterol levels in WT, but not in LOX-1tg mice. HCD did not alter AngII responsiveness or AT1 expression in both vascular beds; however, AngII responsiveness and AT1 expression were lower in aortas from LOX-1tg compared with WT mice. In aortas from WT-CD mice, acute oxLDL exposure induced AT1-mediated vasoconstriction via LOX-1. HCD impaired endothelium-dependent vasodilation and increased superoxide levels in WT aortas, but not uterine arteries. Moreover, in WT-CD mice oxLDL decreased MCh sensitivity in both vascular beds, partially via LOX-1. In summary, HCD impaired pregnancy outcomes and vascular function, and oxLDL-induced LOX-1 activation may contribute to vascular dysfunction via AT1. Our study suggests that LOX-1 could be a potential target to prevent adverse outcomes associated with vascular dysfunction in preeclampsia.

Abstract P080: Mutation Of Lysine 68 To Arginine Mimics Deacetylation Of Mitochondrial Superoxide Dismutase, Protects From Vascular Oxidative Stress And Attenuates Angiotensin II-Induced Hypertension

By recent guidelines, almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs, likely due to mechanisms contributing to blood pressure elevation that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. We found that hypertension is associated with inactivation of key mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to acetylation of lysine residues at the catalytic center. The role of specific SOD2 lysine residues in hypertension, however, has not been defined. We proposed that inactivation of key intrinsic antioxidant, SOD2, in hypertension is linked to acetylation of Lysine 68, and mutation of K68 to Arginine mimics SOD2 deacetylation, inhibits vascular oxidative stress and attenuates angiotensin II-induced hypertension. To test this hypothesis, we have investigated SOD2 acetylation in arterioles from patients with essential hypertension and developed a new deacetylation mimic SOD2 mutant K68R mice (SOD2-K68R). Western blot analysis of arterioles isolated from human mediastinal fat showed 3-fold increase in SOD2 acetylation in hypertensive patients compared with normotensive subjects while SOD2 levels were not affected. To define the functional significance of K68 acetylation we performed studies in vivo in SOD2-K68R mice using angiotensin II model of vascular dysfunction and hypertension. Angiotensin II infusion in wild-type C57Bl/6J mice induced vascular inflammation and oxidative stress, and increased blood pressure to 160 mm Hg. Mutation of Lysine 68 to Arginine in SOD2-K68R mice completely prevented the increase in mitochondrial superoxide and significantly attenuated the angiotensin II induced hypertension (135 mm Hg). Angiotensin II and TNFα co-operatively induce SOD2 acetylation and hypertension. Treatment of wild-type aortas with angiotensin II and TNFα in organoid culture increased mitochondrial superoxide by 2-fold which was completely prevented in aortas isolated from SOD2-K68R mice. These data support an important role of SOD2-K68 acetylation in hypertension, and strategies to reduce mitochondrial acetylation may have therapeutic potential.