Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms.

BACKGROUND: Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. METHODS: TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. RESULTS: Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. CONCLUSIONS: These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.

Sustained depletion of FXIII-A by inducing acquired FXIII-B deficiency.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity.

Adipocyte-specific deletion of HuR induces spontaneous cardiac hypertrophy and fibrosis.

Adipose tissue homeostasis plays a central role in cardiovascular physiology, and the presence of thermogenically active brown adipose tissue (BAT) has recently been associated with cardiometabolic health. We have previously shown that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) reduces BAT-mediated adaptive thermogenesis, and the goal of this work was to identify the cardiovascular impacts of Adipo-HuR-/-. We found that Adipo-HuR-/- mice exhibit a hypercontractile phenotype that is accompanied by increased left ventricle wall thickness and hypertrophic gene expression. Furthermore, hearts from Adipo-HuR-/- mice display increased fibrosis via picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene coexpression network analysis (WGCNA) across both cardiac and adipose tissue to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and cardiac fibrosis. RNA-seq results demonstrated a significant increase in proinflammatory gene expression in both cardiac and subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF-α and IL-6. In addition to inflammation-related genes, WGCNA identified a significant enrichment in extracellular vesicle-mediated transport and exosome-associated genes in scWAT, whose expression most significantly associated with the degree of cardiac fibrosis observed in Adipo-HuR-/- mice, implicating these processes as a likely adipose-to-cardiac paracrine mechanism. These results are significant in that they demonstrate the spontaneous onset of cardiovascular pathology in an adipose tissue-specific gene deletion model and contribute to our understanding of how disruptions in adipose tissue homeostasis may mediate cardiovascular disease.NEW & NOTEWORTHY The presence of functional brown adipose tissue in humans is known to be associated with cardiovascular health. Here, we show that adipocyte-specific deletion of the RNA binding protein HuR, which we have previously shown to reduce BAT-mediated thermogenesis, is sufficient to mediate a spontaneous development of cardiac hypertrophy and fibrosis. These results may have implications on the mechanisms by which BAT function and adipose tissue homeostasis directly mediate cardiovascular disease.

Antithrombotic therapy in abdominal aortic aneurysm: beneficial or detrimental?

Abdominal aortic aneurysm (AAA) is a degenerative vascular pathology resulting in significant morbidity and mortality in older adults due to rupture and sudden death. Despite 150 000 new cases and nearly 15 000 deaths annually, the only approved treatment of AAA is surgical or endovascular intervention when the risk for aortic rupture is increased. The goal of the scientific community is to develop novel pharmaceutical treatment strategies to reduce the need for surgical intervention. Because most clinically relevant AAAs contain a complex structure of fibrin, inflammatory cells, platelets, and red blood cells in the aneurysmal sac known as an intraluminal thrombus (ILT), antithrombotic therapies have emerged as potential pharmaceutical agents for the treatment of AAA progression. However, the efficacy of these treatments has not been shown, and the effects of shrinking the ILT may be as detrimental as they are beneficial. This review discusses the prospect of anticoagulant and antiplatelet (termed collectively as antithrombotic) therapies in AAA. Herein, we discuss the role of the coagulation cascade and platelet activation in human and animal models of AAA, the composition of ILT in AAA, a possible role of the ILT in aneurysm stabilization, and the implications of antithrombotic drugs in AAA treatment.

Dietary Choline Supplementation Attenuates High-Fat-Diet-Induced Hepatocellular Carcinoma in Mice.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death in the world. Choline deficiency has been well studied in the context of liver disease; however, less is known about the effects of choline supplementation in HCC. OBJECTIVE: The objective of this study was to test whether choline supplementation could influence the progression of HCC in a high-fat-diet (HFD)-driven mouse model. METHODS: Four-day-old male C57BL/6J mice were treated with the chemical carcinogen, 7,12-dimethylbenz[a]anthracene, and were randomly assigned at weaning to a cohort fed an HFD (60% kcal fat) or an HFD with supplemental choline (60% kcal fat, 1.2% choline; HFD+C) for 30 wk. Blood was isolated at 15 and 30 wk to measure immune cells by flow cytometry, and glucose-tolerance tests were performed 2 wk prior to killing. Overall tumor burden was quantified, hepatic lipids were measured enzymatically, and phosphatidylcholine species were measured by targeted MS methods. Gene expression and mitochondrial DNA were quantified by quantitative PCR. RESULTS: HFD+C mice exhibited a 50-90% increase in both circulating choline and betaine concentrations in the fed state (P ≤ 0.05). Choline supplementation resulted in a 55% decrease in total tumor numbers, a 67% decrease in tumor surface area, and a 50% decrease in hepatic steatosis after 30 wk of diet (P ≤ 0.05). Choline supplementation increased the abundance of mitochondria and the relative expression of ß-oxidation genes by 21% and ∼75-100%, respectively, in the liver. HFD+C attenuated circulating myeloid-derived suppressor cells at 15 wk of feeding (P ≤ 0.05). CONCLUSIONS: Choline supplementation attenuated HFD-induced HCC and hepatic steatosis in male C57BL/6J mice. These results suggest a therapeutic benefit of choline supplementation in blunting HCC progression.

Roles of Coagulation Proteases and PARs (Protease-Activated Receptors) in Mouse Models of Inflammatory Diseases.

Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.

Tissue-level inflammation and ventricular remodeling in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common cardiac condition caused primarily by sarcomeric protein mutations with several distinct phenotypes, ranging from asymmetric septal hypertrophy, either with or without left ventricular outflow tract obstruction, to moderate left ventricular dilation with or without apical aneurysm formation and marked, end-stage dilation with refractory heart failure. Sudden cardiac death can occur at any stage. The phenotypic variability observed in HCM is the end-result of many factors, including pre-load, after-load, wall stress and myocardial ischemia stemming from microvascular dysfunction and thrombosis; however, tissue level inflammation to include leukocyte-derived extracellular traps consisting of chromatin and histones, apoptosis, proliferation of matrix proteins and impaired or dysfunctional regulatory pathways contribute as well. Our current understanding of the pathobiology, developmental stages, transition from hypertrophy to dilation and natural history of HCM with emphasis on the role of tissue-level inflammation in myocardial fibrosis and ventricular remodeling is summarized.

Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association.

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice.

OBJECTIVE: PAR2 (protease-activated receptor 2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. The objective of this study was to determine the effect of PAR2 deficiency on the development of atherosclerosis. APPROACH AND RESULTS: PAR2 mRNA and protein expression is increased in human carotid artery and mouse aortic arch atheroma versus control carotid and aortic arch arteries, respectively. To determine the effect of PAR2 deficiency on atherosclerosis, male and female low-density lipoprotein receptor-deficient (Ldlr-/-) mice (8-12 weeks old) that were Par2+/+ or Par2-/- were fed a fat- and cholesterol-enriched diet for 12 or 24 weeks. PAR2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root after 12 and 24 weeks. PAR2 deficiency did not alter total plasma cholesterol concentrations or lipoprotein distributions. Bone marrow transplantation showed that PAR2 on nonhematopoietic cells contributed to atherosclerosis. PAR2 deficiency significantly attenuated levels of the chemokines Ccl2 and Cxcl1 in the circulation and macrophage content in atherosclerotic lesions. Mechanistic studies using isolated primary vascular smooth muscle cells showed that PAR2 deficiency is associated with reduced Ccl2 and Cxcl1 mRNA expression and protein release into the supernatant resulting in less monocyte migration. CONCLUSIONS: Our results indicate that PAR2 deficiency is associated with attenuation of atherosclerosis and may reduce lesion progression by blunting Ccl2- and Cxcl1-induced monocyte infiltration.

Tefillin use induces remote ischemic preconditioning pathways in healthy men.

The present study assessed whether tefillin use (tight, nonocclusive, wrapping of the arm) elicits a remote ischemic preconditioning (RIPC)-like effect in subjects with both acute and chronic use. RIPC, created by short bursts of ischemia-reperfusion, has not been successfully taken to the bedside. Several large population studies have found that Orthodox Jewish men (who wear tefillin almost daily) have decreased cardiovascular mortality compared with non-Orthodox counterparts. We hypothesized that tefillin use is a relevant component in triggering a preconditioning effect. Jewish men ( n = 20) were enrolled; 9 men were daily tefillin users (conditioned) and 11 men were nonusers of tefillin as controls (naïve). Subjects were evaluated for adherence to traditional Jewish practice, had vital signs measured, blood drawn for analysis of circulating cytokines and monocyte function, and underwent brachial flow-mediated dilation to evaluate vascular reactivity at baseline (basal) and after 30 min of using tefillin (acute treatment). Under basal conditions, both groups had similar peak systolic velocity (SV), diameter, and flow volume, although the conditioned group had higher SV at 120 s postdeflation ( P = 0.05). Acute tefillin use augmented artery diameter and flow volume in both groups, with conditioned subjects experiencing higher SV than control subjects at 90 and 120 s postdeflation ( P = 0.03 and P = 0.02, respectively). Conditioned subjects had decreased inflammation, monocyte migration and adhesion, and endothelial activation compared with control subjects at baseline. Acute use of tefillin did not significantly alter monocyte function in either group. In this pilot study, acute tefillin use improves vascular function, whereas chronic tefillin use is associated with an anti-inflammatory RIPC-like phenotype. NEW & NOTEWORTHY We hypothesized that tefillin use among Orthodox Jewish men (who practice a nonocclusive leather banding of their nondominant arm) will induce a remote ischemic preconditioning phenotype. Chronic use of tefillin in Orthodox Jewish men was associated with increased systolic velocity and attenuated inflammation and monocyte chemotaxis and adhesion versus Jewish men who do not wear tefillin. Acute use of tefillin in both populations augmented brachial artery diameter and blood flow but not inflammatory profiles compared with baseline.

Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association.

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice.

OBJECTIVE: Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice lacking CDGI or P2Y12 in hematopoietic cells. APPROACH AND RESULTS: Lethally irradiated Ldlr(-/-) mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1(-/-) (cdgI(-/-)), p2y12(-/-), or cdgI(-/-)p2y12(-/-) (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr(-/-) chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr(-/-)/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/p2y12(-/-) chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/ p2y12(-/-) chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr(-/-) chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr(-/-)/DKO chimeras when compared with chimeras lacking only CDGI. CONCLUSIONS: Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr(-/-) mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. METHODS: We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre). RESULTS: Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. CONCLUSIONS: Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.

The antithrombotic effects of statins.

Hypercholesterolemia is considered the primary risk factor for cardiovascular disease. An estimated 200 million prescriptions are issued per year for statins to treat hypercholesterolemia. Importantly, statins have additional beneficial effects independent of their effects on lipids. Recent studies have shown that statins reduce thrombosis via multiple pathways, including inhibiting platelet activation and reducing the pathologic expression of the procoagulant protein tissue factor. Many of the antithrombotic effects of statins are attributed to inhibiting prenylation of RhoA and effects on other intracellular signaling molecules such as NF-κB and KLF2. These antithrombotic activities of statins likely contribute to the ability of statins to reduce the incidence of cardiovascular death.

Platelet Inhibitors Reduce Rupture in a Mouse Model of Established Abdominal Aortic Aneurysm.

OBJECTIVE: Rupture of abdominal aortic aneurysms causes a high morbidity and mortality in the elderly population. Platelet-rich thrombi form on the surface of aneurysms and may contribute to disease progression. In this study, we used a pharmacological approach to examine a role of platelets in established aneurysms induced by angiotensin II infusion into hypercholesterolemic mice. APPROACH AND RESULTS: Administration of the platelet inhibitors aspirin or clopidogrel bisulfate to established abdominal aortic aneurysms dramatically reduced rupture. These platelet inhibitors reduced abdominal aortic platelet and macrophage recruitment resulting in decreased active matrix metalloproteinase-2 and matrix metalloproteinase-9. Platelet inhibitors also resulted in reduced plasma concentrations of platelet factor 4, cytokines, and components of the plasminogen activation system in mice. To determine the validity of these findings in human subjects, a cohort of aneurysm patients were retrospectively analyzed using developed and validated algorithms in the electronic medical record database at Vanderbilt University. Similar to mice, administration of aspirin or P2Y12 inhibitors was associated with reduced death among patients with abdominal aortic aneurysm. CONCLUSIONS: These results suggest that platelets contribute to abdominal aortic aneurysm progression and rupture.

Endothelial Shc regulates arteriogenesis through dual control of arterial specification and inflammation via the notch and nuclear factor-κ-light-chain-enhancer of activated B-cell pathways.

RATIONALE: Arteriogenesis, the shear stress-driven remodeling of collateral arteries, is critical in restoring blood flow to ischemic tissue after a vascular occlusion. Our previous work has shown that the adaptor protein Shc mediates endothelial responses to shear stress in vitro. OBJECTIVE: To examine the role of the adaptor protein Shc in arteriogenesis and endothelial-dependent responses to shear stress in vivo. METHODS AND RESULTS: Conditional knockout mice in which Shc is deleted from endothelial cells were subjected to femoral artery ligation. Hindlimb perfusion recovery was attenuated in Shc conditional knockout mice compared with littermate controls. Reduced perfusion was associated with blunted collateral remodeling and reduced capillary density. Bone marrow transplantation experiments revealed that endothelial Shc is required for perfusion recovery because loss of Shc in bone marrow-derived hematopoietic cells had no effect on recovery. Mechanistically, Shc deficiency resulted in impaired activation of the nuclear factor κ-light-chain-enhancer of activated B-cell-dependent inflammatory pathway and reduced CD45⁺ cell infiltration. Unexpectedly, Shc was required for arterial specification of the remodeling arteriole by mediating upregulation of the arterial endothelial cell marker ephrinB2 and activation of the Notch pathway. In vitro experiments confirmed that Shc was required for shear stress-induced activation of the Notch pathway and downstream arterial specification through a mechanism that involves upregulation of Notch ligands delta-like 1 and delta-like 4. CONCLUSIONS: Shc mediates activation of 2 key signaling pathways that are critical for inflammation and arterial specification; collectively, these pathways contribute to arteriogenesis and the recovery of blood perfusion.

Microparticles in hemostasis and thrombosis.

Blood contains microparticles (MPs) derived from a variety of cell types, including platelets, monocytes, and endothelial cells. In addition, tumors release MPs into the circulation. MPs are formed from membrane blebs that are released from the cell surface by proteolytic cleavage of the cytoskeleton. All MPs are procoagulant because they provide a membrane surface for the assembly of components of the coagulation protease cascade. Importantly, procoagulant activity is increased by the presence of anionic phospholipids, particularly phosphatidylserine (PS), and the procoagulant protein tissue factor (TF), which is the major cellular activator of the clotting cascade. High levels of platelet-derived PS(+) MPs are present in healthy individuals, whereas the number of TF(+), PS(+) MPs is undetectable or very low. However, levels of PS(+), TF(+) MPs are readily detected in a variety of diseases, and monocytes appear to be the primary cellular source. In cancer, PS(+), TF(+) MPs are derived from tumors and may serve as a useful biomarker to identify patients at risk for venous thrombosis. This review will summarize our current knowledge of the role of procoagulant MPs in hemostasis and thrombosis.

Endothelial cell-specific deficiency of Ang II type 1a receptors attenuates Ang II-induced ascending aortic aneurysms in LDL receptor-/- mice.

RATIONALE: Human studies and mouse models have provided evidence for angiotensin II (Ang II)-based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that Ang II infusion induces aneurysmal pathology in the ascending aorta. OBJECTIVE: The aim of this study was to define the role of angiotensin II type 1a (AT(1a)) receptors and their cellular location in Ang II-induced ascending aortic aneurysms (AAs). METHODS AND RESULTS: Male LDL receptor(-/-) mice were fed a saturated fat-enriched diet for 1 week before osmotic mini-pump implantation and infused with either saline or Ang II (1000 ng/kg per minute) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT(1a) receptor deficiency ablated Ang II-induced ascending AAs (P<0.001). To determine the role of AT(1a) receptors on leukocytes, LDL receptor(-/-)×AT(1a) receptor(+/+) or AT(1a) receptor(-/-) mice were irradiated and repopulated with bone marrow-derived cells isolated from either AT(1a) receptor(+/+) or AT(1a) receptor(-/-) mice. Deficiency of AT(1a) receptors in bone marrow-derived cells had no effect on Ang II-induced ascending AAs. To determine the role of AT(1a) receptors on vascular wall cells, we developed AT(1a) receptor floxed mice with depletion on either smooth muscle or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT(1a) receptor deletion in smooth muscle cells had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology. CONCLUSIONS: Ang II infusion promotes aneurysms in the ascending aorta via stimulation of AT(1a) receptors that are expressed on endothelial cells.

HuR inhibition reduces post-ischemic cardiac remodeling by dampening acute inflammatory gene expression and the innate immune response.

Myocardial ischemia/reperfusion (I/R) injury and the resulting cardiac remodeling is a common cause of heart failure. The RNA binding protein Human Antigen R (HuR) has been previously shown to reduce cardiac remodeling following both I/R and cardiac pressure overload, but the full extent of the HuR-dependent mechanisms within cells of the myocardium have yet to be elucidated. In this study, we applied a novel small molecule inhibitor of HuR to define the functional role of HuR in the acute response to I/R injury and gain a better understanding of the HuR-dependent mechanisms during post-ischemic myocardial remodeling. Our results show an early (two hours post-I/R) increase in HuR activity that is necessary for early inflammatory gene expression by cardiomyocytes in response to I/R. Surprisingly, despite the reductions in early inflammatory gene expression at two hours post-I/R, HuR inhibition has no effect on initial infarct size at 24-hours post-I/R. However, in agreement with previously published work, we do see a reduction in pathological remodeling and preserved cardiac function at two weeks post-I/R upon HuR inhibition. RNA-sequencing analysis of neonatal rat ventricular myocytes (NRVMs) at two hours post-LPS treatment to model damage associated molecular pattern (DAMP)-mediated activation of toll like receptors (TLRs) demonstrates a broad HuR-dependent regulation of pro-inflammatory chemokine and cytokine gene expression in cardiomyocytes. We show that conditioned media from NRVMs pre-treated with HuR inhibitor loses the ability to induce inflammatory gene expression in bone marrow derived macrophages (BMDMs) compared to NRVMs treated with LPS alone. Functionally, HuR inhibition in NRVMs also reduces their ability to induce endocrine migration of peripheral blood monocytes in vitro and reduces post-ischemic macrophage infiltration to the heart in vivo. In summary, these results suggest a HuR-dependent expression of pro-inflammatory gene expression by cardiomyocytes that leads to subsequent monocyte recruitment and macrophage activation in the post-ischemic myocardium.