Aims Abdominal aortic aneurysms (AAA) rupture is a life-threatening event with unclear molecular mechanisms. Our previous work demonstrated elevated levels of the matricellular protein thrombospondin-1 (TSP1, encoded by Thbs1) in human and mouse AAA tissues. Single-cell RNA sequencing analysis identified macrophages, endothelial cells, and smooth muscle cells as the major TSP1-expressing cells in aneurysmal tissues. Global Thbs1 deletion reduces aneurysm formation by inhibiting vascular inflammation. The aim of this study was to investigate how TSP1 deficiency in different cell types affects AAA rupture. Methods and results AAA and rupture were induced by angiotensin II infusion in hypercholesterolemic mice. In global Thbs1 deficient mice, hypercholesterolemia was achieved by crossing them with Apoe knockout mice. To generate cell type-specific TSP1 deficient mice, Thbs1flox/flox mice were crossed with VE-cadherin-Cre, SMMHC-iCreERT2, and Lyz2-Cre mice to target endothelial cells, smooth muscle cells, and myeloid cells, respectively. In these conditional knockout models, hypercholesterolemia was induced via AAV-PCSK9. We found that both global and myeloid-specific Thbs1 deletion increased rupture rate over two-fold, whereas endothelial- or smooth muscle cell-specific deletion had no significant effect. Endothelial-specific Thbs1 deletion reduced aneurysm size in the CaCl₂ model. Single-cell RNA sequencing and histology in myeloid-specific Thbs1 knockout aortas revealed broad suppression of inflammation and extracellular matrix production. Conclusion Myeloid-derived TSP1 plays a critical role in inhibiting aneurysm rupture in mice, likely by promoting matrix repair phenotypes in vascular smooth muscle cells, enhancing vascular wall integrity.

Rationale:Rupture of abdominal aortic aneurysms (AAA) is associated with high mortality. However, the precise molecular and cellular drivers of AAA rupture remain elusive. Our prior study showed that global and myeloid-specific deletion of matricellular protein thrombospondin-1 (TSP1) protects mice from aneurysm formation primarily by inhibiting vascular inflammation. Objective:To investigate the cellular and molecular mechanisms that drive AAA rupture by testing how TSP1 deficiency in different cell populations affects the rupture event. Methods and Results:We deleted TSP1 in endothelial cells and macrophages --- the major TSP1-expressing cells in aneurysmal tissues ---- by crossbreeding Thbs1 flox/flox mice with VE-cadherin Cre and Lyz2-cre mice, respectively. Aortic aneurysm and rupture were induced by angiotensin II in mice with hypercholesterolemia. Myeloid-specific Thbs1 knockout, but not endothelial-specific knockout, increased the rate of lethal aortic rupture by more than 2 folds. Combined analyses of single-cell RNA sequencing and histology showed a unique cellular and molecular signature of the rupture-prone aorta that was characterized by a broad suppression in inflammation and extracellular matrix production. Visium spatial transcriptomic analysis on human AAA tissues showed a correlation between low TSP1 expression and aortic dissection. Conclusions:TSP1 expression by myeloid cells negatively regulates aneurysm rupture, likely through promoting the matrix repair phenotypes of vascular smooth muscle cells thereby increasing the strength of the vascular wall.

Diabetes -associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr - ' - mice on a high -fat, high -sucrose -containing (HFSC) diet identifies a macrophage -enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage -enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNAmRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL -deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet -fed Ldlr - ' - mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro -inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage -enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

Introduction: - Patients with diabetes have a poorer prognosis following myocardial infarction compared to healthy controls. This is partly due to impaired post-ischemic angiogenesis in the diabetic heart. The role of microRNAs (miRs) in angiogenesis is relatively unexplored. miR expression is responsive to stimuli such as hypoxia, metabolic environment, growth factors, etc. Here, we describe how miR-342-3p, a hypoxia-responsive miR, regulates angiogenesis under physiologic and diabetic conditions. Methods: - miR-342-3p or control mimic was injected into the border zone of hearts post-ischemia/reperfusion in male Ldlr -/- mice on a high fat and high sucrose (HFSC) diet. Echocardiography, histology, immunofluorescence, RNA FISH and in vitro assays were used to elucidate the mechanism of action of this miR. Results: - Ejection fraction (EF) and fractional shortening (FS) of miR-342-3p injected hearts were significantly higher by 145% and 173%, respectively, at day 14 compared to controls (p = 0.0204 and p = 0.0095). Capillary density as well as ki67+ endothelial cells were more abundant by 107% and 154%, respectively (p = 0.0371 and p = 0.0242) vs control. RNA sequencing of miR-342-3p overexpressing endothelial cells implicated HGF-MET signaling as one of the top upregulated pathways. Western blots (WB) revealed an increase in MET expression (p = 0.0255) in miR-342-3p overexpressing cells vs control. Decorin (DCN) was identified as a direct target of miR-342-3p and validated by WB and luciferase reporter assays. Hypoxia (2% O 2 ) induced miR-342-3p expression (p = 0.0023) while suppressing DCN levels. However, both high glucose (25 mM) and palmitate (100 μM) treatments blunted the miR-342-3p induction while increasing Decorin levels. Co-IP and microscopy were used to show Decorin-MET co-localization. Lastly, miR-342-3p and DCN were inversely expressed (p = 0.0188 and p = 0.0015 respectively) in diabetic and healthy ECs obtained from patients. Conclusion: - These findings establish a critical role for miR-342-3p in regulating cardiac angiogenesis post-ischemic injury in diabetic mice. Under diabetic conditions, the hypoxia induced expression of miR-342-3p is blunted, leading to Decorin mediated degradation of MET receptors, which impairs angiogenesis.

Diabetes mellitus is a global public health problem whose cases will continue to rise along with the progressive increase in obesity and the aging of the population. People with diabetes exhibit higher risk of cardiovascular complications, especially myocardial infarction (MI). microRNAs (miRNAs) are evolutionary conserved small non-coding RNAs involved in the regulation of biological processes by interfering in gene expression at the post-transcriptional level. Accumulating studies in the last two decades have uncovered the role of stage-specific miRNAs associated with key pathobiological events observed in the hearts of people with diabetes and MI, including cardiomyocyte death, angiogenesis, inflammatory response, myocardial remodeling, and myocardial lipotoxicity. A better understanding of the importance of these miRNAs and their targets may provide novel opportunities for RNA-based therapeutic interventions to address the increased risk of MI in diabetes.

Introduction: Diabetes-associated atherosclerosis is characterized by excessive vascular wall inflammation, including impaired lipid deposition, increased immune cell infiltration and plaque formation in the vessel wall. Macrophages are instrumental for lesion formation, however the mechanisms underlying their impaired functional capacity remain poorly understood in diabetes. Herein, we identify a unique role of a macrophage-enriched lncRNA MERRICAL (Macrophage Enriched lncRNA Regulates Inflammation, Chemotaxis, and AtheroscLerosis) in the development of diabetes-associated atherosclerosis. Methods: Transcriptomic analysis of the aortic intima from Ldlr -/- mice was performed at 4 time points along the continuum of lesion progression and regression under a high fat, high sucrose-containing (HFSC) or chow diet, respectively, to identify differentially expressed lncRNAs. Gain- and loss-of-function cell-based assays, RNA-protein interactions, and in vivo knockdown were performed to uncover the role of a top lncRNA candidate. Results: LncRNA MERRICAL is nuclear localized and increased by 249% in intimal lesions during progression. Transcriptomic analysis of MERRICAL -deficient bone marrow-derived macrophages showed decreased chemokine and inflammatory gene expression for Ccl3, Ccl4, and Il1β, with reduced chemotaxis and inflammatory-associated gene ontology pathways. Gain- and loss-of-function studies demonstrated the role of lncRNA MERRICAL in cis -regulation of its neighboring genes Ccl3 and Ccl4, an effect that is mediated in part via interaction with the histone modification enzyme H3K4me3. Remarkably, systemic knockdown of MERRICAL in HFSC-fed mice strongly reduced lesion formation by 73% in the aortic sinus by inhibiting the pro-inflammatory and Ccl3 - and Ccl4 -associated chemotactic response and decreasing the recruitment of leukocytes into the aortic wall. Conclusions: These findings establish a cis -regulatory mechanism by which a macrophage-specific lncRNA, MERRICAL , potently inhibits pro-inflammatory and chemotactic responses to alleviate lesion progression in diabetes, with implications for a broad range of diabetes-associated vascular disease states.

BACKGROUND AND AIMS:Isolation of cellular constituents from the mouse aorta is commonly used for expression or functional analyses in atherosclerosis research. However, current procedures to isolate primary cells are difficult, inefficient, and require separate mice. RNA extraction from aortic intima and media for transcriptomic analysis is also considered difficult with mixed RNA yields. To address these gaps, we provide: 1) a rapid, efficient protocol to isolate and culture diverse cell types concomitantly from the mouse aorta using immunomagnetic cell isolation; and 2) an optimized aortic intimal peeling technique for efficient RNA isolation from the intima and media. METHODS AND RESULTS:Aortic cells were obtained using an enzymatic solution and different cell types were isolated by magnetic beads conjugated to antibodies targeting endothelial cells (CD31+), leukocytes (CD45+), and fibroblast cells (CD90.2+), and smooth muscle cells were isolated by negative selection. Our protocol allows the isolation of relatively large numbers of cells (10,000 cells per aorta) in a predictable manner with high purity (>90%) verified by cell-marker gene expression, immunofluorescence, and flow cytometry. These cells are all functionally active when grown in cell culture. We also provide a rapid method to collect aortic intima-enriched RNA from Ldlr-/- mice utilizing an intima peeling approach and assess transcriptomic profiling associated with accelerated lesion formation. CONCLUSIONS:This protocol provides an effective means for magnetic bead-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic studies for a range of vascular diseases including atherosclerosis.

BACKGROUND:Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS:TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS:TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS:CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

Introduction: Diabetic patients are more likely to suffer from adverse cardiovascular events such as myocardial infarction (MI) compared to non-diabetics. Angiogenesis is crucial to limiting infarct size. However, this angiogenic response is blunted in patients with diabetes. MicroRNAs (miRNAs) have been shown to affect both physiological and pathological angiogenesis. However, the role of miRNAs in MI-associated angiogenesis in diabetes remains poorly defined. Here, we describe a novel role of miRNA-342-3p in angiogenesis following ischemic injury to the myocardium. Methods: We performed RNA sequencing on cardiac endothelial cells of Ldlr-deficient mice on either chow or high fat sucrose containing (HFSC) diet at 0-, 3-, 7- and 14-days post-MI. Gain and loss of function studies, functional assays, RNA-seq, bioinformatics, RNA-FISH, and light-sheet microscopy were used to ascertain the function and mechanism of this miRNA. Results: miR-342-3p was upregulated in cardiac ECs during the vascular remodeling phase post-MI in chow-fed mice compared to the HFSC-fed group. Overexpression of miR-342-3p promotes the number of endothelial sprouts (140% vs control), cumulative sprout length (311% vs control) and scratch closure (AUC 83% vs control, p <0.0001) in endothelial cells (ECs), whereas its inhibition had anti-angiogenic effects. Capillary density and EC proliferation (ki67 + /CD31 + ) increased by 107% and154% respectively in the border zone of hearts injected with miR-342-3p mimics compared to control in HFSC diet fed Ldlr -/- mice 7 days after permanent LAD ligation. Using a combination of RNA-seq and prediction algorithms, Decorin (DCN), a small Leucine rich protein with anti-angiogenic properties, was identified as a target of miR-342-3p. Western blot analysis confirmed significant knockdown of Decorin protein by 94% in HUVECs transfected with miR-342-3p mimics compared to controls. RNA seq profiling of overexpressing ECs implicated the ID1 signaling pathway. Conclusion: These results identify a novel role for miR-342-3p in the regulation of angiogenesis post-MI in diabetes. miR-342-3p overexpression rescues impaired angiogenesis in diabetic hearts via the suppression of Decorin, providing new targets for therapeutic angiogenesis.

Introduction: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Angiotensin II (Ang II) contributes to vascular disease via promoting T-cell activation and end-organ damage. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. Objective: Kruppel-like Factor 10 is a transcription factor expressed in T cell subsets. We sought to investigate the role of KLF10 in CD4+ T cells in regulating vascular damage in an AngII mouse model. Methods: CD4-targeted KLF10 deficient (TKO) and CD4-Cre (WT) mice were generated and subjected to 28 days of Ang II infusion. Endpoint characterization included fibrotic organ transcriptomic analysis, multi-organ histology, flow cytometry, cytokine analysis, myograph vasoreactivity, and cardiac echocardiography. Results: TKO mice showed enhanced perivascular fibrosis compared to WT mice by histological analysis in the aorta, heart, and kidney. TKO mice had vessels with enhanced vasoconstriction to phenylephrine, hearts with impaired global longitudinal strain by echo, and kidneys with elevated albumin/creatinine ratio. However, no change was found in blood pressure between TKO and WT. Plasma IL-9 and IL-15 were increased in TKO mice. IL-9 mRNA and secreted protein were also increased in activated TKO CD4+ T cells, as well as in Ang II treated WT-CD4+ T cells in vitro . Mechanistic studies revealed that KLF10 regulated Il9 transcription through interaction with promoter region of Il9 by ChIP assay. Notably, injection of anti-IL9 antibody reversed perivascular fibrosis in Ang II infused TKO mice. Conclusion: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for the treatment of vascular or fibrotic diseases.

Introduction: Atherosclerosis is a chronic inflammatory disease of the vascular wall characterized by abnormal lipid deposition, immune cell infiltration, and plaque formation. Isolation of the cellular constituents of the mouse aorta is commonly used for expression or functional analyses in experimental atherosclerosis. Endothelial cell, smooth muscle cell, leukocyte, and fibroblast are four major contributors to the progression of atherosclerosis. However, the isolation and culture of the primary aortic cells is considered difficult, often resulting in low yields. Current procedures are either only focused on isolation of one cell type or required long time for isolation and culture. Objective: To address the gap in experimental atherosclerosis research, we provide a fast and efficient protocol to isolate and culture a diverse range of cell types from the mouse aorta using immunomagnetic cell sorting. Methods and Results: Aortic cells were obtained using an enzymatic solution and different cell types were isolated using magnetic beads conjugated to antibodies targeting endothelial cell (CD31+), leukocyte (CD45+), and fibroblast cell (CD90.2+). Aortic smooth muscle cells were isolated by negatively selection after positive removal of the other cell types. Our protocol allows the isolation of relatively large numbers of cells (approximately 10,000 cells per mouse aorta) in a predictable manner, and the purity of the magnetic beads-selected cells was over 90% which was verified by detection of cell-marker genes expression, immunofluorescence staining, and flow cytometry. We also provide a quick and simple method to collect aortic intima-enriched RNA utilizing an intima peeling approach with Trizol. Conclusion: This protocol provides an effective means for magnetic beads-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic vascular studies including atherosclerosis.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr −/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-β1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600–1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Rationale: Thrombospondin-1 (TSP1) is a matricellular protein with TGFβ dependent and independent functions that has been implicated in various vascular diseases. Our previous work demonstrates tha...

Abdominal aortic aneurysm (AAA) is a common aortic disease with a progressive nature. There is no approved pharmacological treatment to effectively slow aneurysm growth or prevent rupture. Necroptosis is a form of programmed necrosis that is regulated by receptor-interacting protein kinases (RIPs). We have recently demonstrated that the lack of RIP3 in mice prevented aneurysm formation. The goal of the current study is to test whether perturbing necroptosis affects progression of existing aneurysm using the RIP1 inhibitors Necrostatin-1 (Nec-1) and an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s). Seven days after aneurysm induction by elastase perfusion, mice were randomly administered DMSO, Nec-1 (3.2 mg/kg/day) and Nec-1s (1.6 mg/kg/day) via intraperitoneal injection. Upon sacrifice on day 14 postaneurysm induction, the aortic expansion in the Nec-1s group (64.12 ± 4.80%) was significantly smaller than that of the DMSO group (172.80 ± 13.68%) ( P < 0.05). The mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60 ± 10.40%) than the DMSO group, though the difference was not statistically significant ( P = 0.1). Histologically, the aortic structure of Nec-1s-treated mice appeared normal, with continuous and organized elastin laminae and abundant αActin-expressing SMCs. Moreover, Nect-1s treatment diminished macrophage infiltration and MMP9 accumulation and increased aortic levels of tropoelastin and lysyl oxidase. Together, our data suggest that pharmacological inhibition of necroptosis with Nec-1s stabilizes pre-existing aneurysms by diminishing inflammation and promoting connective tissue repair.

Abdominal aortic aneurysm (AAA), the progressive weakening and dilatation most commonly occurred in the infrarenal segment of aorta, is a common aortic disease associated with high lethality. Currently, there is no approved pharmacologic treatment to effectively slow aneurysm growth or prevent rupture. We have recently demonstrated that receptor interacting protein kinase 3 (RIP3), a critical mediator of necroptosis, contributes to smooth muscle depletion and vascular inflammation associated with AAA. Here, we tested whether inhibiting the related necroptotic mediator RIP1, using either necrostatin 1 (Nec-1) or an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s), mitigates aneurysm progression. To induce AAA, male mice (8-2 weeks old) were subjected to intraluminal elastase perfusion for 5 minutes at a constant pressure of 100 mm Hg. We first tested the effects of administering Nec-1 via daily intraperitoneal (IP) injection of 3.2 mg/kg/day. We next studied whether RIP1 inhibition blocks progression of an existing AAA. Necrostatin-1 attenuated smooth muscle necroptosis and, more importantly, prevented aneurysm formation and pathologic features associated with the disease, including elastin fragmentation, macrophage infiltration and smooth muscle cell (SMC) death. Seven days after elastase perfusion, mice were randomly divided into three groups and respectively received daily intraperitoneal injections of DMSO, Nec-1 (3.2 mg/kg/day) or Nec-1s (1.6 mg/kg/day) for the remaining 7 days of the study. The aortic expansion in the Nec-1s group (64.12% ± 4.80%; 0.864% ± 0.032 mm) was significantly smaller than that of DMSO group (172.80% ± 13.68%; 1.408% ± 0.068 mm; P < .05, Kruskal-Wallis nonparametric test). Consistently, aortic tissue from Nec-1s-treated mice had preserved elastic fiber integrity, diminished SMC death, and reduced macrophage infiltration. Although the mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60% ± 10.40%; 1.133 ± 0.052 mm) than the DMSO group, the difference was not statistically significant (P = .1). Our data demonstrate that pharmacologic inhibition of necroptosis not only prevents aneurysm formation but stabilizes pre-existing aneurysms in mice. Necroptotic SMCs may thus serve as a novel drug target for treatment of AAA.

Rationale: Depletion of medial smooth muscle cell (SMC) is a major pathological characteristic of abdominal aortic aneurysm (AAA), although the mechanism by which these cells are eliminated remains incompletely understood. We reasoned that necroptosis, a recently described form of necrosis mediated by receptor-interacting protein kinase 3 (RIP3), may contribute to AAA pathology through the induction of SMC death and the significant production of inflammatory cytokines. Objective: To test the hypothesis that RIP3-mediated necroptosis is actively involved in aneurysm pathogenesis. Methods and Results: RIP3 and RIP1 levels were found to be elevated in human AAAs, most noticeably in SMCs. Elevations of RIP3 and SMC necrosis were also observed in the elastase-induced mouse model of AAAs. Deletion of one or both copies of Rip3 prevented AAA formation. By transplanting Rip3 +/− aortae to Rip3 +/+ mice, we demonstrated that reduced Rip3 expression in arterial wall was the primary cause of aneurysm resistance. In vitro, adenoviral overexpression of RIP3 was sufficient to trigger SMC necroptosis. Protein kinase C-delta contributed to tumor necrosis factor-α–induced SMC necroptosis by regulating Rip3 expression. Furthermore, Rip3 deficiency impaired tumor necrosis factor-α–induced inflammatory gene expression in aortic SMCs, which was at least in part because of attenuation of p65 Ser536 phosphorylation. In vivo, the lack of RIP3 diminished activation of p65 in SMCs, implicating a necrosis independent function of RIP3 in aneurysms. Conclusions: Enhanced RIP3 signaling in aneurysmal tissues contributes to AAA progression by causing SMC necroptosis, as well as stimulating vascular inflammation, and therefore may serve as a novel therapeutic target for AAA treatment.

Objective: Abdominal aortic aneurysm (AAA) is a common vascular disease with a progressive nature. Currently, no pharmacological treatment is approved to effectively slow aneurysm growth or prevent rupture. We have recently demonstrated that receptor interacting protein kinase 3 (RIP3), a critical mediator of necroptosis, contributes to smooth muscle depletion and vascular inflammation associated with AAA. In this study, we tested the hypothesis that inhibition of necroptosis may mitigate aneurysm progression using Necrostatin-1 (Nec-1) or an optimized form of Nec-1 called Nec-1s (7-Cl-O-Nec-1), known inhibitors of another necroptosis mediator RIP1. Approach and Results: Using elastase perfusion model, we first demonstrated that Nec-1 attenuated aneurysm formation when administered daily by intraperitoneal (IP) injection started 30 min before aneurysm induction. Nec-1 also profoundly reduced elastin fragmentation, macrophage infiltration and SMC necrosis after elastase perfusion. To test whether RIP1 inhibitors can inhibit AAA progression, we randomly divided mice to four groups 7 days after elastase perfusion when aortic dilatation is small but significant. Group 1 was sacrificed to obtain a baseline aortic dilatation, while Group 2, 3, and 4 received daily IP injections of DMSO, Nec-1 (3.2 mg/kg/day) or Nec-1s (1.6mg/kg/day), respectively. 14 days after perfusion, mice in Group 2 displayed larger aneurysmal expansion as compared to Group 1 ( P <0.05), a reflection of aneurysm growth. In contrast, mice in Group 3 and 4 showed similar aortic dilatations compared to mice in Group 1 ( P >0.05), indicating insignificant aneurysmal growth. Furthermore, real-time PCR and histological analyses demonstrated that RIP1 inhibition significantly reduced aortic accumulation of proinflammatory cytokines and inflammatory cell infiltration. Conclusions: Taken together, our study suggests that necroptosis may serve as a therapeutic target for AAAs. Pharmacological inhibition of RIP1 kinase activity prevented aneurysm formation and stabilized pre-existing aneurysms in mice.