Application Of Elastase Research Articles

Abstract 3003: Ketogenic Diet Limits Experimental Abdominal Aortic Aneurysms

Background: Intermittent fasting and ketogenic diet may improve cardiovascular health. We studied the impact of ketogenic dietary intervention on experimental abdominal aortic aneurysms (AAAs). Methods: AAAs were induced in male mice via topical abluminal porcine pancreatic elastase (PPE) application at laparotomy in C57BL/6J, and 28 day administration of exogenous angiotensin (Ang) II in apolipoprotein E deficient strains, respectively. As feeding regimens, mice were fed standard or ketogenic diets 3 days prior to or 3 days following, AAA induction. Influence on AAA development was assessed by serial transabdominal ultrasonography and histopathologic analysis at sacrifice. Results: Ketogenic diet promotes cardiovascular health in part by increasing ketone body production. The Figure summarizes the influence of ketogenic diet on experimental AAAs in two distinct models. Following PPE application, subsequent aortic diameter enlargement was significantly reduced in mice receiving ketogenic vs. standard diets at 7 and 14 days regardless of specific feeding regimen. On histologic analysis, ketogenic diet feeding resulted in improved aortic medial elastin and smooth muscle cell retention, reduced mural macrophage, CD4 + and CD8 + T and B cell infiltration, and attenuated mural angiogenesis. In the Ang II infusion model, ketogenic diet limited Ang II-induced aortic diameter expansion, lowered AAA incidence and reduced AAA severity. Conclusion: Ketogenic dietary intervention suppressed experimental AAA progression in complementary murine modeling systems. Already popular clinically, this dietary modification may prove effective in limiting clinical AAA disease progression as well as other cardiovascular disease endpoints.

Abstract 1015: Knockout Of P27 Kip1 Accelerates Topical Elastase/3-aminopropionitrile Fumarate Salt-induced Abdominal Aortic Aneurysm Rupture In Mice

Background: Abdominal aortic aneurysms (AAAs) are multifactorial diseases, and unknown genetic factors could contribute to aneurysms. The cell cycle inhibitor CDKN1B gene, also known as p27 Kip1 (p27), affects the arterial response to injury. Previous studies have shown that the loss of p27 exacerbates atherosclerosis progression by increasing vascular smooth muscle cells and macrophages. To assess the role of p27 in AAA development, another form of arterial response to inflammatory injury, we conducted experiments on the AAA model using p27 knockout (p27 -/- ) and wild-type (WT) mice. Methods: AAAs were induced in male and female p27 -/- and WT (C57BL/6J) mice by topically applying porcine pancreatic elastase and administrating 0.2% 3-aminopropionitrile fumarate salt (BAPN) in the drinking water. Topical elastase application was divided into active and heat-inactivated elastase groups. The aortic diameter was evaluated by ultrasound examination preoperatively and every 7 days postoperatively ( Fig. A ). Surviving mice were sacrificed after the final ultrasound evaluation, and aortic samples were collected for histological analysis. Results: Fig. B shows the aortic diameter change evaluated by ultrasound examination. AAA rupture occurred in one male p27-/- mice (33%, 1 of 3) and two female p27-/- mice (40%, 2 of 5), but in no WT mice of either gender. Macrophages were assessed using anti-F4/80 antibody. In the elastase-active group, both male and female p27 -/- mice had a significantly higher accumulation of macrophage than WT mice (P<0.01 and P<0.01, respectively), but there was no significant staining of F4/80 in the elastase-inactive group ( Fig. C and D ). Conclusions: Knockout of p27 exacerbated aneurysm rupture in a topical elastase/BAPN-induced AAA model and was associated with increased macrophage infiltration.

Abstract 2128: Hepcidin Loss Demonstrates Sex Dependent Differences In Aortic Aneurysm Size

Background: Studies suggest circulating iron levels play a role in the pathophysiology of abdominal aortic aneurysms (AAAs). Low iron levels lead to medial degeneration and VSMC dysfunction while elevated iron levels promote oxidative stress. Hepcidin is a major iron regulatory hormone that binds ferroportin to reduce serum iron levels, and its removal in VSMCs has been shown to exacerbate AAAs; however, the role of sex in this mechanism remains understudied. The goal of the current study is to investigate the potential role of sex in iron metabolism in AAAs. Methods: Male and female C57BL/6 wild-type (WT/WT), heterozygous hepcidin knock-out (WT/KO) and hepcidin KO (KO/KO) underwent peri-adventitial application of porcine pancreatic elastase on the infrarenal aorta (n=10-19/group). At day 14, the aortas were measured using video micrometry and harvested for immunohistochemistry. In separate studies, male and female hepcidin WT/WT and KO/KO underwent elastase application and systemic b-aminopropionitrile (BAPN, 0.02%) administration and were harvested at 28 days. Finally, male and female LysM Cre Ferroportin flx/flx mice and their controls underwent elastase application and harvested at 14 days (n=5-10/group). Results: Hepcidin WT/KO and KO/KO male mice had dose dependent protection of aneurysm formation compared with controls (WT/WT: 145.5±33.8% vs WT/KO: 110.7±34.8% vs KO/KO: 53.7±13.7%, p=0.0001 by ANOVA). Female Hepcidin WT/KO and KO/KO mice exhibited increased AAA size compared with their controls (KO/KO: 119.0±23.5% vs WT/KO: 101.6 ±28.6% vs WT/WT: 71.2±22.0%, p =.0011 by ANOVA). In the elastase + BAPN model, Hepcidin KO/KO male mice had less aortic dilation compared to controls (KO/KO: 196.0 ±31.9% vs WT/WT: 476.8±82.2%, p =.0002 by Mann-Whitney) while female KO/KO mice exhibited greater aortic dilation (WT/WT: 186.7±26.0% vs. KO/KO: 406.9±62.2%, p=0.006). Male LysM Cre Ferroportin flx/flx mice had decreased aortic dilation (WT/WT: 132.5±19.3% vs flx/flx: 53.5±7.8%, p=0.001). Conclusion: Elimination of hepcidin in AAA formation exhibited protective effects in male and deleterious effects in female mice. This data suggests targeting of hepcidin using drug-based therapies could have gender-specific clinical implications.

Suppression of Inflammation-induced Abdominal Aortic Aneurysm Formation by Induction of Elastin Tolerance

Abdominal aortic aneurysm (AAA) is a vascular disease process whereby the aorta expands to apoint where rupture may occur. This serious condition is diagnosed in approximately 200,000 people in the United States per year and accounts for over 15,000 deaths annually. The only medical intervention proven to reduce the risk of AAA rupture is surgical repair; however, such repair is associated with high risk of death, reduced quality of life, and high expense. AAA is caused by the weakening of the artery wall due to inflammation-induced destruction of its structural components. Our clinical data shows increased levels of circulating elastin degradation products in patients with AAA, especially smokers, compared to risk factor matched controls. This observation led us to hypothesize that an immune reaction to elastin fragments initiates the inflammatory cascade in the aorta that leads to AAA formation. To test this hypothesis, C57BL/6 mice were injected with poly(lactide-co-glycolide) nanoparticle-encapsulated IL-10 to induce immune tolerance or nanoparticle-encapsulated control ovalbumin. Injection of elastin fragments was performed 7 days later to induce an immune response. AAA of the infrarenal aorta was induced by topical application of elastase during laparotomy procedure 14 days after nanoparticle injection. Aorta diameter was measured 16 days post-operatively with Microfil. Immunologic changes were evaluated by cytokine analysis, Tr1/Th17 cell ratio inperipheral blood, and splenic Th17 and Tr1 response to elastin. Based on prior work, we expect that induction of elastin tolerance using poly(lactide-co-glycolide) nanoparticle-encapsulated IL-10 will suppress abdominal aortic aneurysm expansion and promote an anti-inflammatoryenvironment characterized by increased Tr1/Th17 cell ratio, increased levels of anti-inflammatory cytokines, and decreased pro-inflammatory cytokine expression.

Loss of TIMP3, but not TIMP4, exacerbates thoracic and abdominal aortic aneurysm

AimsAorta exhibits regional heterogeneity (structural and functional), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. Region-dependent functions have been reported for TIMP3 and TIMP4 in vascular pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. Methods & resultsTAA or AAA was induced in male and female mice lacking TIMP3 (Timp3−/−), TIMP4 (Timp4−/−) or in wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females, with a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA, while increased inflammation was detected in the media post-AAA, but in the adventitia post-TAA. Timp3−/− mice showed impaired intimal barrier integrity post-AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in AAA versus TAA. Severity of TAA and AAA in Timp4−/− mice was similar to WT mice. In vitro, Timp3 knockdown more severely compromised the permeability of human aortic EC monolayer compared to Timp4 knockdown or the control group. In aneurysmal aorta specimens from patients, TIMP3 expression decreased in the media in AAA, and in adventitial in TAA specimens, consistent with the impact of its loss in AAA versus TAA in mice. ConclusionTIMP3 loss exacerbates inflammation, adverse remodeling and aortic dilation, but triggers different patterns of remodeling in AAA versus TAA, and through different mechanisms.

PRDM16 deficiency in vascular smooth muscle cells aggravates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is usually asymptomatic until life-threatening complications occur, predominantly involving aortic rupture. Currently, no drug-based treatments are available, primarily due to limited understanding of AAA pathogenesis. The transcriptional regulator PR domain-containing protein 16 (PRDM16) is highly expressed in the aorta, but its functions in the aorta are largely unknown. By RNA-seq analysis, we found that vascular smooth muscle cell-specific (VSMC-specific) Prdm16-knockout (Prdm16SMKO) mice already showed extensive changes in the expression of genes associated with extracellular matrix (ECM) remodeling and inflammation in the abdominal aorta under normal housing conditions without any pathological stimuli. Human AAA lesions displayed lower PRDM16 expression. Periadventitial elastase application to the suprarenal region of the abdominal aorta aggravated AAA formation in Prdm16SMKO mice. During AAA development, VSMCs undergo apoptosis because of both intrinsic and environmental changes, including inflammation and ECM remodeling. Prdm16 deficiency promoted inflammation and apoptosis in VSMCs. A disintegrin and metalloproteinase 12 (ADAM12) is a gelatinase that can degrade various ECMs. We found that ADAM12 is a target of transcriptional repression by PRDM16. Adam12 knockdown reversed VSMC apoptosis induced by Prdm16 deficiency. Our study demonstrated that PRDM16 deficiency in VSMCs promoted ADAM12 expression and aggravates AAA formation, which may provide potential targets for AAA treatment.

Abstract 267: Vascular Smooth Muscle Cell-specific Deletion Of Protein Kinase R-like Endoplasmic Reticulum Kinase Attenuates Microbiome-enhanced Abdominal Aortic Aneurysm

Background: We have previously linked the microbiome-derived metabolite trimethylamine N-oxide (TMAO) to abdominal aortic aneurysm (AAA) in both associative human and mechanistic mouse studies. TMAO is reported to bind protein kinase R-like endoplasmic reticulum kinase (PERK), resulting in selective activation of the unfolded protein response (UPR). Concordantly, our previous RNA sequencing results indicate that TMAO augments PERK-mediated UPR pathways in the aneurysm wall. Thus, the current study will determine the functional role of TMAO-mediated PERK activation in aortic VSMCs to AAA pathogenesis. Methods & Results: Ldlr -/- mice with VSMC-specific deletion of Perk were produced by breeding hemizygous male mice expressing Cre under the control of the Transgelin promoter ( B6.Cg-TG(Tagln-cre)1Her/J , Jackson Labs) to female Perk floxed mice ( Eif2ak3 tm1.2Drc /J , Jackson Labs). Specific deletion of PERK in VSMCs was validated by western blot. Male mice were subjected to angiotensin II (AngII) infusion via osmotic mini pump (1,000 ng/kg/min; 28 days), while female mice underwent laparotomy and application of topical elastase (10 mg/mL porcine pancreatic elastase 5 minutes) to induce AAA. To augment circulating TMAO levels, mice were fed a high choline diet (0.2% total cholesterol + 1.2% choline supplementation) for one week prior to and throughout the study. Male mice with VSMC specific PERK knockout demonstrated significantly reduced AngII-induced aortic dilation (Cre+: 0.90 ± 0.048 mm; Cre-: 1.57 ± 0.156 mm; P = 0.001) and rupture induced death relative to control. Similarly, female mice with VSMC specific PERK knockout displayed significantly reduced aortic diameter (Cre+: 1.17 ± 0.069 mm; Cre-: 2.65 ± 0.432 mm; P = 0.002) and rupture induced mortality as compared to control. Additionally, male and female mice with VSMC specific PERK knockout had significantly greater Type I collagen and reduced macrophage accumulation compared to control. Conclusions: These results indicate that VSMC specific knockout of PERK blunts AAA formation in two independent murine models. Further studies are needed to elucidate the mechanism of PERK mediated ER stress in AAA, which may reveal novel therapeutic targets for AAA treatment.

Loss of tissue inhibitor of proteinase 3 (TIMP3), but not TIMP4, worsens abdominal and thoracic aortic aneurysm

Background: Aorta exhibits regional heterogeneity (structural and mechanical), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. TIMP3 and TIMP4 have shown region-dependent functions in aortic pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. Methods and Results: TAA or AAA was induced in male or female mice lacking TIMP3 ( Timp3 -/- ), TIMP4 ( Timp4 -/- ) or wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females. Timp3 -/- mice exhibited a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA whereas inflammation increased in the media post-AAA, but in the adventitia post-TAA. Timp3 -/- mice showed impaired intimal barrier integrity ( in vivo) following AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in TAA versus AAA. Timp4 -/- mice responded similarly to TAA and AAA induction as the WT. In vitro, Timp3 knockdown in human aortic EC more severely compromised the EC monolayer permeability compared to Timp4 knockdown and control groups following elastase treatment. In human aorta specimens, TIMP3 showed a region-specific expression pattern in TAA versus AAA consistent with the impact of its loss in the impaired intimal barrier in AAA, and the adventitial region in TAA. Conclusions: TIMP3 plays a protective role in both AAA and TAA, whereas loss of TIMP4 has minimal impact on the severity of aneurysm formation. TIMP3 loss in the aneurysmal aorta could be responsible for the inflammation, adverse remodeling and aortic dilation in AAA and TAA, through different mechanisms. Support: CIHR, Heart and Stroke. CIHR, Heart and Stroke. This is the full abstract presented at the American Physiology Summit 2023 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 155: Platelet-derived Transforming Growth Factor-β1 Ameliorates AAA in a Murine Model

Background: Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model. Methods and Results: Ldlr -/- mice with platelet-specific deletion of Tgfβ1 were produced by breeding hemizygous male mice expressing Cre under the control of the Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male TGFβ1-Pf4 Cre+ mice (n=8) had significantly increased abdominal aortic diameters compared to Male TGFβ1-Pf4 Cre- mice (n=7) ( Cre- : 1.70 ± 0.12 mm; Cre+ : 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male TGFβ1-Pf4 Cre+ mice died of AAA rupture compared to 0% in the TGFβ1-Pf4 Cre- cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr 2 ), Ldlr -/- /βr2 Flox /PF4 Cre+ and Cre- mice were created, similarly to the previously described Tgfβ1 mice. Male βr2-Pf4 Cre+ (n=7) had increased aortic diameter compared to βr2-Pf4 Cre- (n=6) ( Cre- : 1.70± 0.20 mm; Cre+ : 2.45 ± 0.38 mm; P < 0.138). Additionally, female βr2-Pf4 Cre+ (n=9) also had significantly larger aortic diameters compared to βr2-Pf4 Cre- (n = 5) ( Cre- : 1.27± 0.04 mm; Cre+ : 1.90 ± 0.23 mm; P < 0.01). Conclusions: Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.

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.

PRDM16 Deficiency in Vascular Smooth Muscle Cells Aggravates Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is usually asymptomatic until severe complications with high risk of lethality occur, predominantly involving aortic rupture. Currently, no drug-based treatments are available, primarily due to limited understanding of AAA pathogenesis. PR domain-containing protein 16 (PRDM16) is a transcriptional regulator that governs gene expression by cooperating with other transcription factors. It is highly expressed in the aorta, but its functions in the aortic tissue and AAA pathology are largely unknown. In this study, we investigated the roles of PRDM16 in vascular smooth muscle cells (VSMCs). By RNA-seq analysis, we found that VSMCs-specific Prdm16 knockout mice ( Prdm16 SMKO) already showed extensive changes in the expression of genes associated with extracellular matrix (ECM) remodeling and inflammation in the abdominal aorta under normal housing conditions without any pathological stimuli. We found that human AAA lesions displayed decreased PRDM16 expression in the aortic medial layer. Periadventitial elastase application to the suprarenal region of the abdominal aorta aggravated AAA formation in Prdm16 SMKO mice. During AAA development, VSMCs undergo apoptosis because of both intrinsic and environmental changes including inflammation and ECM remodeling. Prdm16 deficiency promoted inflammation and apoptosis in VSMCs. A disintegrin and metalloproteinase 12 (ADAM12) is a gelatinase which can degrade various ECM. We found that ADAM12 is a target of transcriptional repression by PRDM16. Adam12 knockdown reversed VSMC apoptosis induced by Prdm16 deficiency. Our study demonstrated that PRDM16 deficiency in VSMCs promoted ADAM12 expression and aggravates AAA formation, which may provide potential targets for AAA treatment. This work was partially supported by National Institutes of Health grants HL151524 (L. Chang) This is the full abstract presented at the American Physiology Summit 2023 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.

Murine model of elastase-induced proximal thoracic aortic aneurysm through a midline incision in the anterior neck.

This study was performed to develop a murine model of elastase-induced proximal thoracic aortic aneurysms (PTAAs). The ascending thoracic aorta and aortic arch of adult C57BL/6J male mice were exposed through a midline incision in the anterior neck, followed by peri-adventitial elastase or saline application. The maximal ascending thoracic aorta diameter was measured with high-resolution micro-ultrasound. Twenty-eight days after the operation, the aortas were harvested and analyzed by histopathological examination and qualitative polymerase chain reaction to determine the basic characteristics of the aneurysmal lesions. Fourteen days after the operation, the dilation rate (mean ± standard error) in the 10-min elastase application group (n = 10, 71.44 ± 10.45%) or 5-min application group (n = 9, 42.67 ± 3.72%) were significantly higher than that in the saline application group (n = 9, 7.37 ± 0.94%, P < 0.001 for both). Histopathological examination revealed aortic wall thickening, degradation of elastin fibers, loss of smooth muscle cells, more vasa vasorum, enhanced extracellular matrix degradation, augmented collagen synthesis, upregulated apoptosis and proliferation capacity of smooth muscle cells, and increased macrophages and CD4+ T cells infiltration in the PTAA lesions. Qualitative analyses indicated higher expression of the proinflammatory markers, matrix metalloproteinase-2 and -9 as well as Collagen III, Collagen I in the PTAAs than in the controls. We established a novel in vivo mouse model of PTAAs through a midline incision in the anterior neck by peri-adventitial application of elastase. This model may facilitate research into the pathogenesis of PTAA formation and the treatment strategy for this devastating disease.

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.

Colchicine Does Not Reduce Abdominal Aortic Aneurysm Growth in a Mouse Model.

Background and Aims The nacht domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is upregulated in human abdominal aortic aneurysm (AAA), but its pathogenic role is unclear. The aims of this study were firstly to examine whether the inflammasome was upregulated in a mouse model of AAA and secondly to test whether the inflammasome inhibitor colchicine limited AAA growth. Methods AAA was induced in eight-week-old male C57BL6/J mice with topical application of elastase to the infrarenal aorta and oral 3-aminopropionitrile (E-BAPN). For aim one, inflammasome activation, abdominal aortic diameter, and rupture were compared between mice with AAA and sham controls. For aim two, 3 weeks after AAA induction, mice were randomly allocated to receive colchicine (n = 28, 0.2 mg/kg/d) or vehicle control (n = 29). The primary outcome was the rate of maximum aortic diameter increase measured by ultrasound over 13 weeks. Results There was upregulation of NLRP3 markers interleukin- (IL-) 1β (median, IQR; 15.67, 7.11-22.60 pg/mg protein versus 6.87, 4.54-11.60 pg/mg protein, p = .048) and caspase-1 (109, 83-155 relative luminosity units (RLU) versus 45, 38-65 RLU, p < .001) in AAA samples compared to controls. Aortic diameter increase over 80 days (mean difference, MD, 4.3 mm, 95% CI 3.3, 5.3, p < .001) was significantly greater in mice in which aneurysms were induced compared to sham controls. Colchicine did not significantly limit aortic diameter increase over 80 days (MD -0.1 mm, 95% CI -1.1, 0.86, p = .922). Conclusions The inflammasome was activated in this mouse model of AAA; however, daily oral administration of colchicine did not limit AAA growth.

3D Ultrasound Measurements Are Highly Sensitive to Monitor Formation and Progression of Abdominal Aortic Aneurysms in Mouse Models.

BackgroundAvailable mouse models for abdominal aortic aneurysms (AAAs) differ substantially in the applied triggers, associated pathomechanisms and rate of vessel expansion. While maximum aortic diameter (determined after aneurysm excision or by 2D ultrasound) is commonly applied to document aneurysm development, we evaluated the sensitivity and reproducibility of 3D ultrasound to monitor aneurysm growth in four distinct mouse models of AAA.MethodsThe models included angiotensin-II infusion in ApoE deficient mice, topical elastase application on aortas in C57BL/6J mice (with or without oral administration of β-aminoproprionitrile) and intraluminal elastase perfusion in C57BL/6J mice. AAA development was monitored using semi-automated 3D ultrasound for aortic volume calculation over 12 mm length and assessment of maximum aortic diameter.ResultsWhile the models differed substantially in the time course of aneurysm development, 3D ultrasound measurements (volume and diameter) proved highly reproducible with concordance correlation coefficients > 0.93 and variations below 9% between two independent observers. Except for the elastase perfusion model where aorta expansion was lowest and best detected by diameter increase, all other models showed high sensitivity of absolute volume and diameter measurements in monitoring AAA formation and progression by 3D ultrasound. When compared to standard 2D ultrasound, the 3D derived parameters generally reached the highest effect size.ConclusionThis study has yielded novel information on the robustness and limitations of semi-automated 3D ultrasound analysis and provided the first direct comparison of aortic volume increase over time in four widely applied mouse models of AAA. While 3D ultrasound generally proved highly sensitive in detecting early AAA formation, the 3D based volume analysis was found inferior to maximum diameter assessment in the elastase perfusion model where the extent of inflicted local injury is determined by individual anatomical features.

The role of elastin on the mechanical properties of the anterior leaflet in porcine tricuspid valves.

Elastin is present in the extracellular matrix (ECM) of connective tissues, and its mechanical properties are well documented. In Marfan syndrome, however, the inability to properly code for the protein fibrillin-1 prematurely leads to the degradation and loss of elastin fiber integrity in the ECM. In this study, the role of elastin in the ECM of the anterior leaflet of the tricuspid valve was investigated by examining the biomechanical behavior of porcine leaflets before and after the application of the enzyme elastase. Five loading protocols were applied to the leaflet specimens in two groups (elastase-treated and control samples). The mechanical response following elastase application yielded a significantly stiffer material in both the radial and circumferential directions. At a physiological level of stress (85 kPa), the elastase group had an average strain of 26.21% and 6.32% in the radial and circumferential directions, respectively, at baseline prior to elastase application. Following elastase treatment, the average strain was 5.28% and 0.97% in the radial and circumferential directions, respectively. No statistically significant change was found in the control group following sham treatment with phosphate-buffered saline (PBS). Two-photon microscopy images confirmed that after the removal of elastin, the collagen fibers displayed a loss of undulation. With a significant reduction in radial compliance, the ability to withstand physiological loads may be compromised. As such, an extracellular matrix that is structurally deficient in elastin may hinder normal tricuspid valve function.

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abstract 14198: Peptide-siRNA Nanoparticles Targeting NF-kB Subunit P50 Suppress Experimental Abdominal Aortic Aneurysm Progression and Prevent Rupture

Rationale: Abdominal aortic aneurysm (AAA) is a progressive vascular condition associated with high risk of mortality due to rupture if left untreated. AAA is an inflammatory process with excessive local production of extracellular matrix degrading enzymes, leading to dilatation, and rupture of the abdominal aorta. We posit that targeting NF-κB, a signaling pathway that controls inflammation, will halt AAA progression and prevent rupture. Methods and Results: In an elastase-induced AAA model we observe that NF-κB activation increases progressively post-elastase perfusion. Unexpectedly, we found that AAA progression is marked by predominant nuclear localization of the NF-κB p50 subunit at the exclusion of p65. Using the amphipathic peptide p5RHH to form nanocomplexes with siRNA, we show that NF-κB p50, but not p65 knockdown mitigates small AAA progression. Since elastase-induced AAA is self-limiting, we turn to an AAA model by topical application of elastase combined with TGF-β inhibition, which leads to rapid aortic diameter expansion and rupture in 50% of experimental animals. Systemic delivery of p5RHH-siRNA nanoparticles suppressing p50 protect 100% of male mice from rupture (and sudden death) in this model but does not significantly mitigate aneurysm expansion. In both models, knockdown of p50 is accompanied by significant decrease in inflammatory infiltrate, release of various cytokines, inducible nitric oxide synthase expression, and cell apoptosis. Conclusions: These results identify NF-κB pathway p50 subunit as a critical key modulator of the inflammatory process in AAA and p50 represents a potential therapeutic target in the medical management of AAA to prevents expansion and rupture. Figure 1. (A) Elastase + anti-TGF-β AAA model. (B) Aortic rupture with hematoma (arrow). (C) Survival curve (n = 6-10 mice per treatment group). p50 knockdown prevents rupture and death (C) but not AAA expansion (D). * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001. inact.=inactive.

Evaluation of Circulating Endothelial Progenitor Cells in Abdominal Aortic Aneurysms after Endovascular Aneurysm Repair.

Background and ObjectivesCirculating endothelial progenitor cells (EPCs) participate in vascular repair and predict cardiovascular outcomes. The aim of this study was to investigate the correlation between EPCs and abdominal aortic aneurysms (AAAs).Methods and ResultsPatients (age 67±9.41 years) suffering from AAAs (aortic diameters 58.09±11.24 mm) were prospectively enrolled in this study. All patients received endovascular aneurysm repair (EVAR). Blood samples were taken preoperatively and 14 days after surgery from patients with aortic aneurysms. Samples were also obtained from age-matched control subjects. Circulating EPCs were defined as those cells that were double positive for CD34 and CD309. Rat models of AAA formation were generated by the peri-adventitial elastase application of either saline solution (control; n=10), or porcine pancreatic elastase (PPE; n=14). The aortas were analyzed using an ultrasonic video system and immunohistochemistry. The levels of CD34＋/CD309＋ cells in the peripheral blood mononuclear cell populations were measured by flow cytometry. The baseline numbers of circulating EPCs (CD34＋/CD309＋) in the peripheral blood were significantly smaller in AAA patients compared with control subjects. The number of EPCs doubled by the 14th day after EVAR. A total of 78.57% of rats in the PPE group (11/14) formed AAAs (dilation ratio >150%). The numbers of EPCs from defined AAA rats were significantly decreased compared with the control group.ConclusionsEPC levels may be useful for monitoring abdominal aorta aneurysms and rise after EVAR in patients with aortic aneurysms, and might contribute to the rapid endothelialization of vessels.