Wild-type Aortas Research Articles

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

Regulation of the Phenotypic Switch of Vascular Smooth Muscle Cells by Adiponectin

Vascular smooth muscle cells (VSMCs) play an essential role in the development and progression of atherosclerosis. The phenotypic switch from contractile to synthetic cells induces excess proliferation and migration of VSMCs into the intima contributing to plaque formation. Adiponectin (Adpn) is an adipokine that regulates glucose metabolism through AMPK and plays beneficial roles in atherosclerosis. We hypothesize that Adpn promotes the contractile phenotype of VSMC reducing proliferation and migration of VSMCs, a process that is protective against plaque formation.Differentiation, proliferation, and signaling through AMPK‐dependent pathways were assessed in VSMCs isolated from aortas of wild type (WT) and Adpn−/− mice. Proliferation markers, including cyclin D1, were increased in Adpn−/− cells, and AMPK activity and differentiation markers were reduced. Cells were analyzed for matrix metalloproteinases (MMPs) which are responsible for extracellular matrix (ECM) remodeling, a contributing factor to the VSMC phenotypic switch. We found that MMP protein expression and activity, measured by zymography, were reduced in Adpn−/− cells. To test whether the phenotypic switch is regulated by AMPK, we treated cells with Metformin, an AMPK activator, or AdipoRon, an activator of Adpn receptors. Metformin and AdipoRon reduced Cyclin D1 and MAPK signaling in Adpn−/− cells, suggesting that AMPK prevents VSMC proliferation; however, none of the treatments yielded an effect on markers of differentiation.These data illustrated that Adpn regulates proliferation through an AMPK‐dependent signaling pathway, while regulating differentiation by an AMPK‐independent mechanism. The role of MMPs in VSMC differentiation in response to Adpn remains to be elucidated.

Inhibition of Marfan‐associated Aortic Root Dilation by Angiotensin II Receptor Blockers May Be Independent of Blood Pressure Lowering

BackgroundMarfan syndrome (MFS) is a connective tissue disorder resulting in accelerated vascular aging, aortic root widening and aneurysm if unmanaged. Despite encouraging pre‐clinical data, recent clinical trials have shown underwhelming efficacy of anti‐hypertensive angiotensin II receptor type 1 (ATR1) blocker (ARB) losartan in MFS patients. We have recently reported that losartan mediates its anti‐aortic root remodeling effects in blood pressure (BP)‐ and ATR1‐independent fashions, and instead acts by improving endothelial function and protective endothelial nitric oxide (NO) synthase‐mediated NO release. Considering the unique mechanism of action of losartan in MFS, whether other ARBs can provide greater protection against MFS aortic root widening is unknown.ObjectiveHere we compare the efficacy of other ARBs previously linked to endothelial function activity with losartan at blocking aortic root widening in MFS mice.Methods and ResultsAortic root dilation, aortic vessel remodeling, BP, and aortic contractility were compared between MFS and wild‐type (WT) mice after an 18‐week treatment with losartan, telmisartan, valsartan, or vehicle. Losartan and telmisartan doses were titrated to give similar hemodynamic effects in vivo, resulting in a 20–30% decrease in BP in both groups, while valsartan was chosen at a non‐BP lowering dose. Untreated MFS mice showed increased aortic root widening compared to WT controls (1.84 ± 0.05mm vs 1.52 ± 0.03mm; p<0.05) as assessed by high frequency ultrasound imaging. Despite differences in BP lowering, all ARBs significantly reduced aortic root widening in the MFS group by 10–16% (p<0.05). Histological assessment revealed a 26–37% (p<0.05) reduction in MFS aortic root medial thickening, and a 50% reduction in elastic fiber fragmentation (p<0.0001) in all treatment groups. Using ex vivo small chamber myography, we showed that all ARBs reduced phenylephrine‐induced aortic contractility by 60–80% (p<0.05) in both MFS and WT aortas in a NO‐dependent fashion.ConclusionThis is the first animal study to compare the effects of ARBs other than losartan in MFS. Despite differential BP effects, telmisartan and valsartan were effective at blocking MFS aortic root widening and activating endothelial function. This suggests BP lowering to be of minor therapeutic value in MFS management and thus aortic root protection could be achieved with lesser side effects for MFS patients. Future clinical trials should focus on other ARBs than losartan for the management of MFS.Support or Funding InformationSupported by CIHR, HSFC, BCKDF, Marfan Foundation, SPH Foundation and Rare Disease Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dietary fats modify vascular fat composition, eNOS localization within lipid rafts and vascular function in obesity.

We tested whether dietary fatty acids alter membrane composition shifting localization of signaling pathways within caveolae to determine their role in vascular function. Wild type (WT) and caveolin‐1‐deficient mice (cav‐1 KO), required for vascular caveolae formation, were fed low fat (LF), high saturated fat (HF, 60% kcal from lard), or high‐fat diet with 50:50 lard and n‐3 polyunsaturated fatty acid‐enriched menhaden oil (MO). HF and MO increased body weight and fat in WT but had less effect in cav‐1 KO. MO increased unsaturated fatty acids and the unsaturation index of aorta from WT and cav‐1 KO. In LF WT aorta, endothelial nitric oxide synthase (eNOS) was localized to cav‐1‐enriched low‐density fractions which shifted to actin‐enriched high‐density fractions with acetylcholine (ACh). HF and MO shifted eNOS to high‐density fractions in WT aorta which was not affected by ACh. In cav‐1 KO aorta, eNOS was localized in low‐density non‐caveolar fractions but not shifted by ACh or diet. Inducible NOS and cyclooxygenase 1/2 were not localized in low‐density fractions or affected by diet, ACh or genotype. ACh‐induced dilation of gracilis arteries from HF WT was similar to dilation in LF but the NOS component was reduced. In WT and cav‐1 KO, dilation to ACh was enhanced by MO through increased role for NOS and cyclooxygenase. We conclude that dietary fats affect vascular fatty acid composition and membrane localization of eNOS but the contribution of eNOS and cyclooxygenase in ACh‐mediated vascular responses is independent of lipid rafts.

Abstract 313: Loss of Contractile Smooth Muscle Cell Phenotype in a Mouse Model of Marfan Syndrome

Objectives: Thoracic aortic aneurysm (TAA) is a chronic vascular disease and the 15th leading cause of death in the US. TAA can result from a genetic disorder such as Marfan syndrome (MFS). MFS patients typically have fibrillin-1 deficiency, causing elastic fiber defects in the aortic media that initiate a series of cellular events. We sought to determine the association of smooth muscle cell (SMC) conversion from contractile to synthetic phenotype with changes in extracellular matrix (ECM), medial cell density, and arterial biomechanics. Methods: A mouse model of MFS (Fbn1 C1039G/+, Het) was used to study progression of TAA and cellular events over a 3-12 month time course. Small animal ultrasound was used to image the ascending aorta and to measure diameters at several positions. Immunohistochemistry (IHC) was performed on proximal aorta sections with smooth muscle-myosin heavy chain (SM-MHC) and SM alpha actin as markers for contractile SMCs. Second harmonic generation (SHG) microscopy was used to quantitate aortic medial collagen. Results: Ultrasound data show that aortic diameter significantly increases with time in Het mice. There is a statistically significant difference between genotypes independent of time. IHC images demonstrate that while SM-MHC expression in wild type (WT) mice remains nearly constant, in Het mice at 3 months the expression is comparable to that of WT mice but decreases at 6 months and starts to recover to the WT level by 12 months. SHG images show a time dependent increase in medial collagen expression in Het mice vs. WT. These results suggest loss of contractile SMCs in the media and remodeling of ECM. Conclusion: Our results to date demonstrate loss of contractile SMCs and increased medial collagen deposition in the proximal aorta during TAA progression. Loss of contractile cells together with changes in ECM are expected to alter aortic biomechanics. To quantify these alterations, we are currently studying active and passive biomechanics of ascending aortas of WT and Het mice.

Inhibition of acid sphingomyelinase‐mediated autophagic flux contributes to amitriptyline‐induced perturbation of tube formation in murine endothelial cells

Amitriptyline is an inhibitor of lysosomal acid sphingomyelinase (gene symbol Smpd1) and a well‐known tricyclic anti‐depressant with cardiovascular side effects. We previously reported that loss of acid sphingomyelinase function leads to defective autophagic flux and enhanced dedifferentiation in vascular smooth muscle cells. However, it remains unknown whether amitriptyline has any cardiovascular side effects on the angiogenic capability of vascular endothelial cells and whether such effect is associated with impaired autophagy signaling pathways. Using an aortic ring assay, we demonstrated that the wild‐type aortas treated with amitriptyline or Smpd1+/− aortas exhibited decreased endothelial tube outgrowth from small slices of aorta embedded in Matrigel indicating attenuated ex vivo angiogenesis. Similarly, amitriptyline or Smpd1 gene silencing perturbed the tube formation capability of cultured mouse microvascular endothelial cells (MVECs). The inhibitory effect of amitriptyline on angiogenesis in MVECs was correlated with decreased activation of angiogenic signaling (phosphorylation of eNOS, Akt, and Erk1/2) and reduced endothelial cell proliferation. Mechanistically, amitriptyline alone had no effect on autophagy induction in MVECs but inhibited autophagic flux as shown by increased expression of LC3B and p62. Blockade of autophagic flux by lysosome function inhibitor bafilomycin or chloroquine mimics the effects of amitriptyline on angiogenic signaling, proliferation, and tube formation in MVECs. Collectively, these data suggest that defective acid sphingomyelinase‐mediated autophagic flux contributes to the cardiovascular side effects and toxicity of amitriptyline on proliferation and angiogenic capability of vascular endothelial cells.Support or Funding InformationNational Heart, Lung, and Blood Institute grants (HL122769, HL122937)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

180 The aorta can act as site of t cell priming and promotes a local cd4+ adaptive immune response in early stage atherosclerosis in apolipoprotein-e-/- mice

Artery tertiary lymphoid organs (ATLOs) in the adventitia adjacent to intimal plaque in aortas from aged apolipoprotein-E (apoE)-/- mice have recently been shown by us to orchestrate the aortic immune...

Cyclic Strain and Hypertension Increase Osteopontin Expression in the Aorta.

Hypertension has a direct impact on vascular hypertrophy and is a known risk factor for the development of atherosclerosis. Osteopontin (OPN) has emerged as an important protein mediator of inflammation and remodeling of large arteries. However, its role and mechanism of regulation in the setting of hypertension is still unknown. Our objectives for this study were therefore to investigate the role of OPN in hypertension-induced vascular remodeling and inflammation. OPN Knockout (KO) and wild type (WT) mice were made hypertensive with angiotensin II (Ang II) infusion for seven days. We observed that OPN KO aortas were protected against Ang II-induced medial hypertrophy and inflammation, despite comparable increases in systolic blood pressure (SBP) in both groups. OPN expression was increased in WT aortas from hypertensive mice (induced by either Ang II or norepinephrine). OPN expression was increased in aortic smooth muscle cells (SMCs) subjected to cyclic mechanical strain suggesting that mechanical deformation of the aortic wall is responsible in part for the increased OPN expression induced by hypertension. Finally, we utilized hypertensive transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat) mice to determine the role of H2O2 in mediating hypertension-induced increases in OPN expression. We also found that the hypertension-induced increase in OPN expression was inhibited in transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat) mice, suggesting that H2O2, plays a vital role in mediating the hypertension-induced increase in OPN expression. Taken together, these results define a potentially important role for OPN in the pathophysiology of hypertension.

The P2Y2 receptor mediates uptake of matrix-retained and aggregated low density lipoprotein in primary vascular smooth muscle cells

Background and aimsThe internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs. MethodsPrimary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R−/− mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). ResultsP2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R−/− VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R−/− VSMCs versus cells transfected with the mutant P2Y2R. ConclusionsTogether, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A.

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy.

ObjectivesMarfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis.MethodsWe performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM).ResultsIHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space.ConclusionsMurine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.

Abstract 13212: The Role of Macrophage STAT3 Signaling in Pathogenesis of Aortic Dissection

Aortic dissection (AD) is a fatal disease caused by the disruption of the intimomedial layer. Recent studies showed that IL-6, a STAT3-activating cytokine, and macrophages play important roles in AD. However, it is unknown whether or how activation of macrophage STAT3 is involved in AD pathogenesis. To answer this question, we examined both mouse AD model and human AD tissue. We used macrophage-specific knockout of SOCS3 (mSOCS3-KO), a negative regulator of STAT3, to specifically sensitize macrophages to STAT3 activation, and compared them with wild type (WT) mice. We created a mouse model of aortic stiffening by periaortic CaCl2 treatment of abdominal aorta, and infused them with angiotensin II (Ca+AngII), which augmented hemodynamic stress to aortic walls. Both WT and mSOCS3-KO showed microscopic injuries in aorta with equal frequencies (40%) 1 week after Ca+AngII. Whereas WT showed fibrotic repair of the injury, mSOCS3-KO failed to show such reparative response and developed large AD in 6 weeks. We analyzed the aorta 1 week after the Ca+AngII stimulation, focusing on the changes in aorta preceding AD development. Transcriptome analysis showed higher activation of cell cycle followed by higher proinflammatory response in mSOCS3-KO than in WT aorta before AD development. Ki67 staining and BrdU uptake study confirmed the proliferative response of inflammatory cells in aorta. Flowcytometric analysis showed preferential differentiation of mSOCS3-KO macrophages to proinflammatory M1 with reduction in reparative M2 population (M1:M2 = 2:1), while WT macrophages differentiated equally to M1 and M2. Immunofluorescence study of human AD tissue showed the infiltration of CD68-positive macrophages in adventitia and outer media at the site of dissection and surrounding intact aortic walls. The infiltrating cells showed high phospho- (active) STAT3 and Ki67. Interestingly, STAT3 was less active in tissue with fibrotic changes, suggesting the negative association of STAT3 activation and tissue repair. Therefore, macrophage STAT3 activation resulted in higher proliferation and M1 differentiation, which is likely to promote AD development. Over activation of STAT3 in macrophages may be a therapeutic target to mitigate the destructive process in AD.

Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2 mutations.

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations.

Type 2 diabetes aggravates Alzheimer's disease-associated vascular alterations of the aorta in mice.

Vascular risk factors are associated with a higher incidence of dementia. In fact, diabetes mellitus is considered a main risk factor for Alzheimer's disease (AD) and both diseases are characterized by vascular dysfunction. However, the underlying mechanisms remain largely unknown. Here, the effects of high-sucrose-induced type 2 diabetes (T2D) in the aorta of wild type (WT) and triple-transgenic AD (3xTg-AD) mice were investigated. 3xTg-AD mice showed a significant decrease in body weight and an increase in postprandial glycemia, glycated hemoglobin (HbA1c), and vascular nitrotyrosine, superoxide anion (O2•-), receptor for the advanced glycation end products (RAGE) protein, and monocyte chemoattractant protein-1 (MCP-1) levels when compared to WT mice. High-sucrose intake caused a significant increase in body weight, postprandial glycemia, HbA1c, triglycerides, plasma vascular cell adhesion molecule 1 (VCAM-1), and vascular nitrotyrosine, O2•-, RAGE, and MCP-1 levels in both WT and 3xTg-AD mice when compared to the respective control group. Also, a significant decrease in nitric oxide-dependent vasorelaxation was observed in 3xTg-AD and sucrose-treated WT mice. In conclusion, AD and T2D promote similar vascular dysfunction of the aorta, this effect being associated with elevated oxidative and nitrosative stress and inflammation. Also, AD-associated vascular alterations are potentiated by T2D. These findings support the idea that metabolic alterations predispose to the onset and progression of dementia.

Maintenance of GLUT4 expression in smooth muscle prevents hypertension-induced changes in vascular reactivity.

Previous studies have shown that expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice and that total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity in hypertension. To demonstrate that the effect of GLUT4 overexpression on vascular responses is dependent on vascular smooth muscle GLUT4 rather than on some systemic effect we developed and tested smooth-muscle-specific GLUT4 transgenic mice (SMG4). When made hypertensive with angiotensin II, both wild-type and SMG4 mice exhibited similarly increased systolic blood pressure. Responsiveness to phenylephrine, serotonin, and prostaglandin F2α was significantly increased in endothelium-intact aortic rings from hypertensive wild-type mice but not in aortae of SMG4 mice. Inhibition of Rho-kinase equally reduced serotonin-stimulated contractility in aortae of hypertensive wild-type and SMG4-mice. In addition, acetylcholine-stimulated relaxation was significantly decreased in aortic rings of hypertensive wild-type mice, but not in rings of SMG4 mice. Inhibition of either prostacylin receptors or cyclooxygenase-2 reduced relaxation in rings of hypertensive SMG4 mice. Inhibition of cyclooxygenase-2 had no effect on relaxation in rings of hypertensive wild-type mice. Cyclooxygenase-2 protein expression was decreased in hypertensive wild-type aortae but not in hypertensive SMG4 aortae compared to nonhypertensive controls. Our results demonstrate that smooth muscle expression of GLUT4 exerts a major effect on smooth muscle contractile responses and endothelium-dependent vasorelaxation and that normal expression of GLUT4 in vascular smooth muscle is required for appropriate smooth muscle and endothelial responses.

Role of p38 mitogen-activated protein kinase in vascular endothelial aging: interaction with Arginase-II and S6K1 signaling pathway.

p38 mitogen-activated protein kinase (p38) regulates cellular senescence and senescence-associated secretory phenotype (SASP), i.e., secretion of cytokines and/or chemokines. Previous work showed that augmented arginase-II (Arg-II) and S6K1 interact with each other to promote endothelial senescence through uncoupling of endothelial nitric oxide synthase (eNOS). Here we demonstrate eNOS-uncoupling, augmented expression/secretion of IL-6 and IL-8, elevation of p38 activation and Arg-II levels in senescent endothelial cells. Silencing Arg-II or p38α in senescent cells recouples eNOS and inhibits IL-6 and IL-8 secretion. Overexpression of Arg-II in young endothelial cells causes eNOS-uncoupling and enhances IL-6 and IL-8 expression/secretion, which is prevented by p38 inhibition or by antioxidant. Moreover, p38 activation and expression of IL-6 and KC (the murine IL-8 homologue) are increased in the heart and/or aortas of wild type (WT) old mice, which is abolished in mice with Arg-II gene deficiency (Arg-II-/-). In addition, inhibition of p38 in the old WT mice recouples eNOS function and reduces IL-6 and KC expression in the aortas and heart. Silencing Arg-II or p38a or S6K1 inhibits each other in senescence endothelial cells. Thus, Arg-II, p38, and S6K1 form a positive circuit which regulates endothelial senescence and cardiovascular aging.

Abstract 16789: Loss of Endothelial Barrier in Fibrillin-1-Deficient Marfan Mice (mgR/mgR) Results in Severe Inflammation After Adenoviral Gene Therapy

Introduction: Nowadays surgery still remains option of choice for therapy of aneurysm in Marfan syndrome. Aortic tissue of Marfan patients shows elastolysis enhanced by proteolytic activity of Matrix Metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of Tissue Inhibitor of MMP-1 (TIMP-1) in aortic grafts of Fibrillin-1-deficient Marfan mice (mgR/mgR) in order to reduce aortic elastolysis. Methods: We performed heterotopic infrarenal transplantation of thoracic aorta in female mice (n=35, n=7/group). MgR/mgR and wild-type (WT) aorta (C57BL/6) was either incubated ex vivo with adenoviral vector coding for human TIMP-1 or ß-Galactosidase (5x10^9pfu/ml) or as control with culture medium. 30 days after surgery overexpression of transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography (ISZ). Histologic staining was performed to outline elastin breaks, signs of inflammation and Neointimal Index (NI). Endothelial barrier was evaluated with the use of transmission electron microscope (TEM) and perfusion of fluorescent albumin. Results: IHC and ISZ revealed sufficient expression of transgene. Severe intimal hyperplasia and distinctive signs of cellular inflammation were solely observed in mgR/mgR aorta after adenoviral transduction (NI: ß-Gal 0.23; TIMP-1 0.43), but not in WT aorta (NI: Native 0.01; TIMP-1 0.00). Compared to native mgR/mgR and TIMP-1 treated WT aorta, NI in TIMP-1 treated mgR/mgR aorta was highly significantly larger (p=0.001; p=0.001). Native mgR/mgR grafts presented with severe elastolysis compared to WT (p=0.001). Regardless of the vector transduced elastolysis in mgR/mgR aorta after adenoviral gene therapy was more pronounced compared to WT aorta (ß-Gal p=0.001; TIMP-1 p=0.001). Albumin diffusion through the endothelial barrier was increased in native mgR/mgR aorta compared to native WT aorta (p=0.037). TEM analysis of mgR/mgR aorta displayed reconstruction of the basement membrane and basolateral space. Conclusions: Murine Marfan aortic grafts developed severe inflammation after adenoviral exposure. Fibrillin-1-deficiency is associated with dysfunction of endothelial barrier which could be a target for alternative therapies in Marfan syndrome.

Abstract 431: Lipoprotein Metabolism and Transendothelial Apolipoprotein A-I Transport in Mice With an Endothelium-Specific Knockout of ATP Binding Cassette Transporter A1

Low plasma levels of high density lipoprotein (HDL) cholesterol as well as apolipoprotein A-I (ApoA-I) are associated with increased risk of coronary heart disease. HDL and ApoA-I exert multiple potentially anti-atherogenic activities. Several of them the including stimulation of cholesterol efflux from macrophage foam cells for reverse cholesterol transport are exerted in the vascular wall rather than in the plasma compartment. HDL or apoA-I must hence pass the endothelium to enter and leave the arterial wall. We previously demonstrated in vitro that the transendothelial transport of ApoA-I involves the interaction with the ATP-binding cassette transporter (ABC) A1. To investigate the physiological relevance of transendothelial ApoA-I transport and its regulation by ABCA1, we generated an endothelium-specific ABCA1 knock-out mouse model (ABCA1e-/-) by expressing the Cre recombinase under the control of the Ve Cadherin gene in floxed ABCA1 -/- mice. Under both chow diet and a high fat/high cholesterol diet for 16 weeks the ABCA1e-/- mice did not differ from wild type mice by plasma levels of cholesterol or triglycerides or lipoprotein profiles. After injection of radio-iodinated ApoA-I, we found no difference in the decay of plasma radioactivity and the accumulation of radiolabel in different organs except the aorta. Compared to the aortas of wild type mice, aortas of ABCA1e-/- mice showed a significantly reduced enrichment of the radiolabel. Aortas of ABCA1e-/- mice also showed a slightly decreased enrichment of radiolabeled HDL but no difference in the enrichment of radiolabeled LDL or albumin. An ApoA-I mutant with defective ABCA1 interaction showed enhanced catabolism in both wild type and ABCA1e-/- mice as compared to wild type apoA-I. However the ABCA1-defective ApoA-I mutant did not accumulate differently in the aortas of wild type and ABCA1e-/- mice. Taken together the data support our concept and provide first in vivo evidence that transendothelial ApoA-I transport is a specific process modulated by ABCA1.

Abstract 114: Macrophages Mediate the Ability of the Receptor for Advanced Glycation End Products to Prevent Formation of Abdominal Aortic Aneurysm in a Murine Model

INTRODUCTION: The pathophysiology of AAAs is multifactorial, involving chronic inflammation, increased MMP expression/activity, and medial elastic fiber destruction. RAGE transduces the biological impact of ligand families including AGEs, S100, and HMGB1 to upregulate inflammatory and tissue injury-provoking genes including MMPs. We previously identified increased RAGE expression in human AAAs and have shown that the absence of RAGE, whether by genetic deletion or pharmacologic blockade with sRAGE, prevents AAA formation in a mouse model. We sought to characterize the cellular mediators of this effect. METHODS: C57BL/6 wild-type (WT) and RAGE null (KO) mice underwent transient elastase perfusion to induce AAAs. Mice were sacrificed at day 14 and flow cytometry performed to characterize differences in activation (CD69+) of lymphocytes (CD4/8), neutrophils (GR1+/CD11b+), macrophages (F480), NK cells (NK1.1) (n=24/group). Immunohistochemistry was performed for Mac-3 and Arg 1 (M2 marker) or Arg 2 (M1 marker). Primary BMDMs were isolated and skewed towards M1 (LPS/IFN ¡) or M2 (LPS, IL-4, IL-13) with the addition of S100b or HMGB1 to determine the ligands' effect upon macrophage skewing by ELISA. RESULTS: Flow cytometry revealed increased aortic expression of activated CD8+ cells (p=0.002), macrophages (p-0.002), and NK cells (p=0.03). No differences were noted in cellular activation from spleens from the same animals. Immunohistochemistry revealed increased macrophage expression in WT vs KO aortae, highest at day 5 (p=0.04). Arg 1 and 2 expression on macrophages was elevated in WT vs KO animals (p=0.03). Skewing BMDMs from WT and KO towards M1 led to elevated TNFα with addition of either S100b (p=0.02) or HMGB1 (p=0.03). Skewing BMDMs towards M2 led to elevated IL-10 in KO vs WT cells with the addition of S100b (p=0.01). HMGB1 has no effect upon skewing towards M2. CONCLUSIONS: The effect of RAGE in the development of murine AAA appears to be mediated by macrophages. Differential macrophage skewing towards an M1 by both S100b and HMGB1 likely plays a role in this effect. Skewing towards M2 is mediated by S100b and not HMGB1. Further studies will seek to identify at which stage in AAA formation activation of the RAGE-ligand axis becomes functionally important.

Lp(a)/apo(a) Modulate MMP-9 Activation and Neutrophil Cytokines in Vivo in Inflammation to Regulate Leukocyte Recruitment

Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases, but the mechanism is unclear. The pathogenic risk of Lp(a) is associated with elevated plasma concentration, small isoforms of apolipoprotein [apo(a)], the unique apolipoprotein of Lp(a), and a mimic of plasminogen. Inflammation is associated with both the initiation and recovery of cardiovascular diseases, and plasminogen plays an important role in leukocyte recruitment. Because Lp(a)/apo(a) is expressed only in primates, transgenic mice were generated, apo(a)tg and Lp(a)tg mice, to determine whether Lp(a)/apo(a) modifies plasminogen-dependent leukocyte recruitment or whether apo(a) has an independent role in vivo. Plasminogen activation was markedly reduced in apo(a)tg and Lp(a)tg mice in both peritonitis and vascular injury inflammatory models, and was sufficient to reduce matrix metalloproteinase-9 activation and macrophage recruitment. Furthermore, neutrophil recruitment and the neutrophil cytokines, CXCL1/CXCL2, were suppressed in apo(a)tg mice in the abdominal aortic aneurysm model. Reconstitution of CXCL1 or CXCL2 restored neutrophil recruitment in apo(a)tg mice. Apo(a) in the plasminogen-deficient background and Lp(a)tg mice were resistant to inhibition of macrophage recruitment that was associated with an increased accumulation of apo(a) in the intimal layer of the vessel wall. These data indicate that, in inflammation, Lp(a)/apo(a) suppresses neutrophil recruitment by plasminogen-independent cytokine inhibition, and Lp(a)/apo(a) inhibits plasminogen activation and regulates matrix metalloproteinase-9 activation and macrophage recruitment.

Isoproterenol Induces Vascular Oxidative Stress and Endothelial Dysfunction via a Giα-Coupled β2-Adrenoceptor Signaling Pathway

ObjectiveSustained β-adrenergic stimulation is a hallmark of sympathetic hyperactivity in cardiovascular diseases. It is associated with oxidative stress and altered vasoconstrictor tone. This study investigated the β-adrenoceptor subtype and the signaling pathways implicated in the vascular effects of β-adrenoceptor overactivation.Methods and ResultsMice lacking the β1- or β2-adrenoceptor subtype (β1KO, β2KO) and wild-type (WT) were treated with isoproterenol (ISO, 15 μg.g−1.day−1, 7 days). ISO significantly enhanced the maximal vasoconstrictor response (Emax) of the aorta to phenylephrine in WT (+34%) and β1KO mice (+35%) but not in β2KO mice. The nitric oxide synthase (NOS) inhibitor L-NAME abolished the differences in phenylephrine response between the groups, suggesting that ISO impaired basal NO availability in the aorta of WT and β1KO mice. Superoxide dismutase (SOD), pertussis toxin (PTx) or PD 98,059 (p-ERK 1/2 inhibitor) incubation reversed the hypercontractility of aortic rings from ISO-treated WT mice; aortic contraction of ISO-treated β2KO mice was not altered. Immunoblotting revealed increased aortic expression of Giα-3 protein (+50%) and phosphorylated ERK1/2 (+90%) and decreased eNOS dimer/monomer ratio in ISO-treated WT mice. ISO enhanced the fluorescence response to dihydroethidium (+100%) in aortas from WT mice, indicating oxidative stress that was normalized by SOD, PTx and L-NAME. The ISO effects were abolished in β2KO mice.ConclusionsThe β2-adrenoceptor/Giα signaling pathway is implicated in the enhanced vasoconstrictor response and eNOS uncoupling-mediated oxidative stress due to ISO treatment. Thus, long-term β2-AR activation might results in endothelial dysfunction.