II-induced Vascular Inflammation Research Articles

Nox1/PAK1 is required for angiotensin II-induced vascular inflammation and abdominal aortic aneurysm formation.

NADPH oxidase 1 (Nox1) is a major isoform of Nox in vascular smooth muscle cells (VSMCs). VSMC activation and extracellular matrix (ECM) remodelling induce abdominal aortic aneurysm (AAA). In this study, we aim to determine the role of Nox1 in the progression of AAA and explore the underling mechanism. ApoE-/-Nox1SMCko mice in which the Nox1 gene was smooth muscle cell (SMC)-specifically deleted in ApoE-/- background, were infused with angiotensin II (Ang II) for 28 days. We found the Nox1 deficiency reduced AAA formation and increased survival compared with ApoE-/-Nox1y/flox mice. Abdominal aortic ROS and monocyts/macrophages were reduced in the ApoE-/-Nox1SMCko mice after Ang II-infusion. The SMC-specific Nox1 deletion caused less elastin fragments and lower matrix metalloproteinase (MMP) activities in the abdominal aorta. Further, we found the Nox1 protein interacted with p21-activated kinase 1 (PAK1) in Ang II-stimulated VSMCs. The PAK1, controlled by Nox1/ROS, promoted VSMC proliferation, migration and differentiation; this is associated with increased activities of vimentin and cofilin, and cytoskeleton modulation. Moreover, we found that the Nox1/PAK1 activated the downstream MAPKs (ERK1/2, p38 and JNKs) and NF-κB, and upregulated Sp1-mediated MMP2 expression upon Ang II-stimulation. Finally, overexpression of PAK1 in the ApoE-/-Nox1SMCko mice increased vascular elastic fibre degradation, pro-inflammatory cytokine expression and AAA incidence. Therefore, we conclude that Nox1, together with PAK1, facilitates Ang II-induced VSMC activation, vascular inflammation and ECM remodelling, and thus potentiates the AAA formation.

MyD88 in myeloid cells promotes angiotensin II-induced vascular inflammation and is associated with all-cause mortality and incident heart failure in humans with arterial hypertension

Abstract Background Angiotensin II (AngII) causes hypertension and promotes vascular accumulation of inflammatory cells. We aimed to determine how myeloid factor of differentiation 88 (MyD88) contributes to vascular dysfunction and arterial hypertension in mice and humans. Methods Male C57BL/6 as well as MyD88-/-, LysMCre/wtMyD88LSL/LSL, LysMCre/wt, TLR2-/-, TLR4-/-, TLR7-/- and TLR9-/- were investigated in the AngII-model of hypertension (1 mg/kg/d for 7days). Biodata analyses were performed in the Gutenberg-Health Study (GHS), a population based cohort. In addition, we performed computational analyses of known hypertension-related genes from genome-wide association studies (GWAS) to investigate whether they preferentially interact with MyD88 in the human protein network. Results MyD88 deficiency attenuated AngII-induced blood pressure increase and endothelial dysfunction in conductance and resistance vessels. Vascular mRNA expression levels of vcam-1, nos2, nox2 were decreased in AngII-infused MyD88-/- mice compared to C57BL/6 controls. Flow cytometric analyses revealed that AngII-induced aortic accumulation of CD11b+Ly6Chi inflammatory monocytes was significantly dampened in MyD88-/- mice. AngII increased mRNA expression of cd62L, cd68 and ccl2, which was blocked in MyD88-/- mice, indicating a role of MyD88 for myeloid cell activation and differentiation. Additionally, aortic interferon-g+ NK cells and mRNA levels of interleukin-12 and interleukin-1b were reduced in AngII-treated MyD88-/- mice. Bone marrow transfer experiments demonstrated a vasoprotective effect of MyD88 deficiency in bone marrow-derived cells, and selective expression of MyD88 in LysMCre/wtMyD88LSL/LSL mice restored AngII-induced pathology, revealing that myelomonocytic cells drives vascular dysfunction in a MyD88-dependent manner. Furthermore, data from 1,274 individuals in the GHS indicated, that MyD88 mRNA expression was associated with all cause mortality and incident chronic heart failure after a median follow-up of 11.6 years. Computational analysis of the human protein interaction network demonstrated that MyD88 is significantly connected to proteins encoded by genetic loci associated with blood pressure traits in multiple GWAS. Conclusion We provide evidence, that MyD88 expressed by myelomonocytic cells promotes AngII-induced vascular dysfunction and arterial hypertension. MyD88 might be used as an inflammatory diagnostic marker for the risk of death in hypertensive patients.

Oviductal Glycoprotein 1 Promotes Hypertension by Inducing Vascular Remodeling Through an Interaction With MYH9.

Hypertension is a common cardiovascular disease that is related to genetic and environmental factors, but its mechanisms remain unclear. DNA methylation, a classic epigenetic modification, not only regulates gene expression but is also susceptible to environmental factors, linking environmental factors to genetic modification. Therefore, globally screening differential genomic DNA methylation in patients with hypertension is important for investigating hypertension mechanisms. Differential genomic DNA methylation in patients with hypertension, individuals with prehypertension, and healthy control individuals was screened using Illumina 450K BeadChip and verified by pyrosequencing. Plasma OVGP1 (oviduct glycoprotein 1) levels were determined using an enzyme-linked immunosorbent assay. Ovgp1 transgenic and knockout mice were generated to analyze the function of OVGP1. The blood pressure levels of the mouse models were measured using the tail-cuff system and radiotelemetry methods. The role of OVGP1 in vascular remodeling was determined by vascular relaxation studies. Protein-protein interactions were investigated using a pull-down/mass spectrometry assay and verified with coimmunoprecipitation and pull-down assays. We found a hypomethylated site at cg20823859 in the promoter region of OVGP1 and plasma OVGP1 levels were significantly increased in patients with hypertension. This finding indicates that OVGP1 is associated with hypertension. In Ovgp1 transgenic mice, OVGP1 overexpression caused an increase in blood pressure, dysfunctional vasoconstriction and vasodilation, remodeling of arterial walls, and increased vascular superoxide stress and inflammation, and these phenomena were exacerbated by angiotensin II infusion. In contrast, OVGP1 deficiency attenuated angiotensin II-induced vascular oxidase stress, inflammation, and collagen deposition. These findings indicate that OVGP1 is a prohypertensive factor that directly promotes vascular remodeling. Pull-down and coimmunoprecipitation assays showed that MYH9 (nonmuscle myosin heavy chain IIA) interacted with OVGP1, whereas inhibition of MYH9 attenuated OVGP1-induced hypertension and vascular remodeling. Hypomethylation at cg20823859 in the promoter region of OVGP1 is associated with hypertension and induces upregulation of OVGP1. The interaction between OVGP1 and MYH9 contributes to vascular remodeling and dysfunction. Therefore, OVGP1 is a prohypertensive factor that promotes vascular remodeling by binding with MYH9.

Neohesperidin Protects Angiotensin II-Induced Hypertension and Vascular Remodeling.

Vascular remodeling due to hypertension is one of the major health challenges facing countries around the world. Neohesperidin, a flavonoid glycoside found in citrus fruits, is an antioxidant. Neohesperidin has been studied for a variety of diseases in addition to hypertension. In this study, angiotensin II was used to induce hypertension in mice (490 ng/kg/min, 14 days). We used H&E, Masson, immunofluorescence, dihydroethidine and qPCR to evaluate the effect of Nehesperidin (50 mg/kg/day, 16 days) on pathological hypertension in mice. Estimating the effect of Nehesperidin on human umbilical vein endothelial cells and vascular smooth muscle cells stimulated by angiotensin II. We found that neohesperidin inhibited angiotensin II-induced hypertension in mice. Neohesperidin reduced angiotensin II-induced vascular hypertrophy, fibrosis, inflammation and oxidative stress in vivo. Neohesperidin inhibited angiotensin II-induced ROS and DNA damage in human umbilical vein endothelial cells. Neohesperidin inhibited angiotensin II-induced migration of vascular smooth muscle cells. The results showed that Nehesperidin acts as an antioxidant and could significantly inhibit angiotensin II induced hypertension and vascular remodeling in vitro and in vivo.

Ascending aortic perivascular adipose tissue inflammation associates with aortic valve disease

BackgroundAscending aortic perivascular adipose tissue (AA-PVAT) mainly comprises brown adipose tissue (BAT), originates from neural crest cells that derive from ectoderm, and plays important role in angiotensin II-induced vascular inflammation and remodeling in mice. However, the characterization and function of human AA-PVAT remains highly unclear. MethodsPatients with coronary artery disease (CAD) (n = 20) and aortic valve disease (AVD) (n = 23) who underwent cardiac surgery consented to take part in transcriptome and histological studies. Paired samples of AA-PVAT, epicardial adipose tissue (EAT), and subcutaneous adipose tissue (SAT) were obtained. RNA sequencing, histological analysis, quantitative reverse transcription polymerase chain reaction and western blot studies were performed on those samples. ResultsHuman AA-PVAT exhibited smaller adipocyte morphology and high expression of brown adipocyte marker. Transcriptome analysis revealed that AA-PVAT showed unique transcriptome characteristics compared with EAT and SAT. While comparing CAD and AVD patients, AA-PVAT exhibited a decreasing brown phenotype and higher inflammatory response in AVD patients. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the differentially expressed genes in AA-PVAT between CAD and AVD patients were involved mainly in the processes of inflammation and metabolism regulation. ConclusionsHuman AA-PVAT is a BAT-like adipose tissue with unique transcriptome characteristics, and exhibits a weakened brown phenotype and an enhanced inflammation response in AVD patients.

Carbon monoxide releasing molecule-2 attenuates angiotensin II-induced IL-6/Jak2/Stat3-associated inflammation by inhibiting NADPH oxidase- and mitochondria-derived ROS in human aortic smooth muscle cells

Abdominal aortic aneurysm (AAA) is a common inflammatory vascular disease. Angiotensin II (Ang II) involves in AAA progression by promoting the proliferation and migration of vascular smooth muscle cells, the degradation of extracellular matrices, and the generation of ROS to lead to vascular inflammation. Carbon monoxide releasing molecule-2 (CORM-2) is known to exert anti-inflammatory and antioxidant activities. However, it remains unclear whether CORM-2 can suppress Ang II-induced vascular inflammation to prevent AAA progression. Therefore, this study aimed to investigate the vasoprotective effects of CORM-2 against Ang II-induced inflammatory responses of human aortic smooth muscle cells (HASMCs) and the underlying mechanisms of those effects. The results showed that Ang II induced inflammatory responses of HASMCs via NADPH oxidase- and mitochondria-derived ROS/NF-κB/IL-6/Jak2/Stat3 pathway which was attenuated by the pretreatment with CORM-2. Additionally, CORM-2 further exhibited anti-inflammatory activities in Ang II-stimulated HASMCs, as indicated by the reduction of monocyte adhesion to HASMCs and migration of HASMCs via the suppression of ICAM-1 and VCAM-1 as well as MMP-2 and MMP-9 levels, respectively. Moreover, Ang II-induced COX-2-mediated PGE2 secretion was also inhibited by the pretreatment with CORM-2. Importantly, our data demonstrated that CORM-2 reversed Ang II-induced IL-6 overexpression dependent on Nrf2 activation and HO-1 expression. Taken together, the present study indicates that CORM-2-induced Nrf2/HO-1 alleviates IL-6/Jak2/Stat3-mediated inflammatory responses to Ang II by inhibiting NADPH oxidase- and mitochondria-derived ROS, suggesting that CORM-2 is a promising pharmacologic candidate to reverse the pathological changes involved in the inflammation of vessel wall for the prevention and treatment of AAA.

Myeloid differentiation 2 deficiency attenuates AngII-induced arterial vascular oxidative stress, inflammation, and remodeling.

Vascular remodeling is a pertinent target for cardiovascular therapy. Vascular smooth muscle cell (VSMC) dysfunction plays a key role in vascular remodeling. Myeloid differentiation 2 (MD2), a cofactor of toll-like receptor 4 (TLR4), is involved in atherosclerotic progress and cardiac remodeling via activation of chronic inflammation. In this study, we explored the role of MD2 in vascular remodeling using an Ang II-induced mouse model and cultured human aortic VSMCs. MD2 deficiency suppressed Ang II-induced vascular fibrosis and phenotypic switching of VSMCs without affecting blood pressure in mice. Mechanistically, MD2 deficiency prevented Ang II-induced expression of inflammatory cytokines and oxidative stress in mice and cultured VSMCs. Furthermore, MD2 deficiency reversed Ang II-activated MAPK signaling and Ang II-downregulated SIRT1 expression. Taken together, MD2 plays a significant role in Ang II-induced vascular oxidative stress, inflammation, and remodeling, indicating that MD2 is a potential therapeutic target for the treatment of vascular remodeling-related cardiovascular diseases.

Myeloid differentiation protein 2 mediates angiotensin II-induced inflammation and mesenchymal transition in vascular endothelium

Angiotensin II (Ang II)-induced vascular inflammation and injury entails endothelial to mesenchymal transition (EndMT). Recent studies have shown that Ang II engages toll-like receptor 4 (TLR4) in the vasculature to mediate adverse effects. Here, we aimed to investigate whether myeloid differentiation protein 2 (MD2), an extracellular molecule indispensable for TLR4 activation, mediates Ang II-induced vascular injury and EndMT. We utilized MD2 knockout mice and wildtype mice treated with a specific MD2 inhibitor to decipher its role in aortas of Ang II-challenged mice. To confirm our results and to provide mechanistic insights, we exposed cultured endothelial cells to Ang II, with or without MD2 silencing. We show that Ang II causes deleterious remodeling and EndMT in aortas of mice within two weeks. These Ang II effects were largely absent in MD2 knockout mice and in wildtype mice treated with a MD2 inhibitor. MD2 silencing in cultured endothelial cells confirmed the essential role of MD2 in Ang II-induced inflammatory factor induction, and EndMT-associated phenotypic change. We also found that Ang II-MD2-EndMT axis involves the activation of nuclear factor-κB. Our studies highlight an essential role of MD2 in Ang II-induced vascular inflammation and EndMT contributing to vascular injury. These results also imply that MD2 may be targeted to dampen inflammatory cardiovascular and EndMT-associated diseases.

TLR2 regulates angiotensin II-induced vascular remodeling and EndMT through NF-κB signaling.

Excessive vascular remodeling has been shown in hypertensive patients. In experimental models of hypertensive vascular injury, such as angiotensin II (Ang II) challenged mice, toll like receptor 2 (TLR2) initiates inflammatory responses. More recently, studies have reported atypical endothelial to mesenchymal transition (EndMT) in vascular injuries and inflammatory conditions. Here, we aimed to investigate whether TLR2 mediates Ang II-induced vascular inflammation and initiates EndMT. In a mouse model of angiotensin II-induced hypertension, we show that aortas exhibit increased medial thickening, fibrosis, and features of EndMT. These alterations were not observed in TLR2 knockout mice in response to Ang II. TLR2 silencing in cultured endothelial cells confirmed the essential role of TLR2 in Ang II-induced inflammatory factor induction, and EndMT-associated phenotypic change. Mechanistically, we found Ang II activates nuclear factor-κB signaling, inducing pro-inflammatory cytokine production, and mediates EndMT in both cultured endothelial cells and in mice. These studies illustrate a novel role of TLR2 in regulating Ang II-induced deleterious vascular remodeling through the induction of EndMT. The studies also suggest that TLR2 may be targeted to alleviate hypertension-associated vascular injury.

PTEN (Phosphatase and Tensin Homolog) Protects Against Ang II (Angiotensin II)-Induced Pathological Vascular Fibrosis and Remodeling-Brief Report.

Pathological vascular remodeling and excessive perivascular fibrosis are major contributors to reduced vessel compliance that exacerbates cardiovascular diseases, for instance, promoting clinically relevant myocardial remodeling. Inflammation plays a significant role in both pathological vascular remodeling and fibrosis. We previously demonstrated that smooth muscle cell-specific PTEN depletion promotes significant vascular fibrosis and accumulation of inflammatory cells. In the current study, we aimed to determine the beneficial role of systemic PTEN elevation on Ang II (angiotensin II)-induced vascular fibrosis and remodeling. Approach and Results: Transgenic mice carrying additional copies of the wild-type Pten gene (super PTEN [sPTEN]) and WT littermates were subjected to Ang II or saline infusion for 14 or 28 days. Compared with WT, Ang II-induced vascular fibrosis was significantly blunted in sPTEN mice, as shown by histochemical stainings and label-free second harmonic generation imaging. The protection against Ang II was recapitulated in sPTEN mice bearing WT bone marrow but not in WT mice reconstituted with sPTEN bone marrow. Ang II-induced elevation of profibrotic and proinflammatory gene expression observed in WT mice was blocked in aortic tissue of sPTEN mice. Immunofluorescent staining and flow cytometry both indicated that perivascular infiltration of T cells and macrophages was significantly inhibited in sPTEN mice. In vitro induction of PTEN expression suppressed Ang II-induced Ccl2 expression in vascular smooth muscle cells. Systemic PTEN elevation mediates protection against Ang II-induced vascular inflammation and fibrosis predominantly through effects in resident vascular cells. Our data highly support that pharmacological upregulation of PTEN could be a novel and viable approach for the treatment of pathological vascular fibrosis.

T Cell-Derived IL-17A Induces Vascular Dysfunction via Perivascular Fibrosis Formation and Dysregulation of ·NO/cGMP Signaling.

Aims The neutrophil recruiting cytokine Interleukin-17A (IL-17A) is a key component in vascular dysfunction and arterial hypertension. Moreover, IL-17A has a central role for the vascular infiltration of myeloid cells into the arterial wall in Angiotensin II-induced vascular inflammation. The intention of our study was to analyze the impact of T cell-derived IL-17A on hypertension, vascular function, and inflammation. Methods and Results Chronic IL-17A overexpression in T cells (CD4-IL-17Aind/+ mice) resulted in elevated reactive oxygen species in the peripheral blood and a significant vascular dysfunction compared to control mice. The vascular dysfunction seen in the CD4-IL-17Aind/+ mice was only accompanied by a modest and nonsignificant accumulation of inflammatory cells within the vessel wall. Therefore, infiltrating myeloid cells did not serve as an explanation of the vascular dysfunction seen in a chronic IL-17A-driven mouse model. In addition to vascular dysfunction, CD4-IL-17Aind/+ mice displayed vascular fibrosis with highly proliferative fibroblasts. This fibroblast proliferation was induced by exposure to IL-17A as confirmed by in vitro experiments with primary murine fibroblastic cells. We also found that the ·NO/cGMP pathway was downregulated in the vasculature of the CD4-IL-17Aind/+ mice, while levels of protein tyrosine kinase 2 (PYK2), an oxidative stress-triggered process associated with T cell activation, were upregulated in the perivascular fat tissue (PVAT). Conclusions Our data demonstrate that T cell-derived IL-17A elicits vascular dysfunction by mediating proliferation of fibroblasts and subsequent vascular fibrosis associated with PYK2 upregulation.

Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Krüppel-like factor (KLF) 15 has emerged as a critical regulator of fibrosis in cardiovascular diseases. However, the precise role that KLF15 and its functional domain played in adventitial inflammation and fibrosis remains unclear. This study aims to investigate the role of the transactivation domain (TAD) of KLF15 in angiotensin II (Ang II)-induced adventitial pathologic changes. KLF15 expression was decreased in the vascular adventitia of Ang II-infused mice (1000 ng/kg/min, 14 d) and in adventitial fibroblasts (AFs) stimulated by Ang II (10-7 M). Adenovirus-mediated KLF15 overexpression normalized Ang II-induced vascular hypertrophy, increased collagen deposition, macrophage infiltration, and CCL2 and VCAM-1 expression. Interestingly, KLF15-ΔTAD (KLF15 with deletion of TAD at amino acids 132-152) overexpression showed no effect on the above pathologic changes. Similarly, perivascularly overexpression of KLF15 but not KLF15-ΔTAD in carotid arteries also attenuated Ang II-induced vascular inflammation and fibrosis. Furthermore, KLF15 overexpression after Ang II infusion rescued Ang II-induced vascular remodeling. CCL2 or VCAM-1-mediated monocyte and macrophage migration or adhesion to AFs in response to Ang II was negatively regulated by KLF15 through TAD. Ang II-enhanced Smad2/3 activation and adventitial migration, proliferation, and differentiation of AFs were suppressed by KLF15 but not KLF15-ΔTAD overexpression. Conversely, small interfering RNA knockdown of KLF15 aggravated Ang II-induced Smad2/3 activation and dysfunction of AFs. Luciferase, coimmunoprecipitation, and chromatin immunoprecipitation assay were used to demonstrate that interaction of KLF15 with Smad2/3 suppressed CCL2 expression through TAD. Mechanistically, activation of Ang II type 1 receptor/phospholipase Cγ 1/ERK1/2 signaling resulted in a decrease of KLF15 expression. In conclusion, these results demonstrate that KLF15 negatively regulates activation of AFs through TAD, which plays an important role in Ang II-induced adventitial inflammation and fibrosis.-Lu, Y.-Y., Li, X.-D., Zhou, H.-D., Shao, S., He, S., Hong, M.-N., Liu, J.-C., Xu, Y.-L., Wu, Y.-J., Zhu, D.-L., Wang, J.-G., Gao, P.-J. Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

HDAC4 regulates vascular inflammation via activation of autophagy.

Angiotensin II (Ang II) causes vascular inflammation, leading to vascular endothelial cell dysfunction, and is associated with the development of cardiovascular diseases. Therefore, interventions in inflammation may contribute to the reduction of cardiovascular diseases. Here, we aim to demonstrate that HDAC4, one of class IIa family histone de-acetylases (HDACs) members, promotes autophagy-dependent vascular inflammation. By loss-of-function approaches, our study provides the first evidence that HDAC4 mediates Ang II-induced vascular inflammation in vitro and in vivo. In response to the Ang II, HDAC4 expression is up-regulated rapidly, with increased autophagic flux and inflammatory mediators in vascular endothelial cells (VECs). In turn, HDAC4 deficiency suppresses activation of autophagy, leading to reduced inflammation in Ang II-induced VECs. Consistently, using autophagy inhibitor or silencing LC3-II also alleviates vascular inflammation. Furthermore, HDAC4 regulates autophagy via facilitating transcription factor forkhead box O3a (FoxO3a) de-acetylation, thereby to increase its transcriptional activity. Loss of HDAC4 in VECs results in inhibition of FoxO3a de-acetylation to block its transcriptional activity, leading to downregulation of the downstream FoxO3a target, and hence reduces autophagy and vascular inflammation. FoxO3a silencing using siRNA approach significantly inhibits activation of autophagy. Finally, knockdown of HDAC4 in Ang II-infused mouse models ameliorates vascular inflammation, suggesting that inhibitor of HDAC4 may be potential therapeutics for vascular diseases associated with inflammation. These results suggest that HDAC4-mediated FoxO3a acetylation regulates Ang II-induced autophagy activation, which in turn plays an essential role in causing vascular inflammation.

Deletion of NF-κB/RelA in Angiotensin II-Sensitive Mesenchymal Cells Blocks Aortic Vascular Inflammation and Abdominal Aortic Aneurysm Formation.

Infusion of angiotensin II (Ang II) induces extracellular matrix remodeling and inflammation resulting in abdominal aortic aneurysms (AAAs) in normolipidemic mice. Although Ang II activates mesenchymal cells in the media and adventitia to become fibrogenic, the sentinel role of this mesenchymal population in modulating the inflammatory response and aneurysms is not known. We test the hypothesis that these fibrogenic mesenchymal cells play a critical role in Ang II-induced aortic wall vascular inflammation and AAA formation. Ang II infusion increased phospho-Ser536-RelA and interleukin (IL)-6 immunostaining in the abdominal aorta. In addition, aortic mRNA transcripts of RelA-dependent cytokines IL-6 and IL-1β were significantly elevated suggesting that Ang II functionally activates RelA signaling. To test the role of mesenchymal RelA in AAA formation, we generated RelA-CKO mice by administering tamoxifen to double transgenic mice harboring RelA-flox alleles and tamoxifen-inducible Col1a2 promoter-driven Cre recombinase (Col1a2-CreERT). Tamoxifen administration to Col1a2-CreERT•mT/mG mice induced Cre expression and RelA depletion in aortic smooth muscle cells and fibroblasts but not in endothelial cells. Infusion of Ang II significantly increased abdominal aortic diameter and the incidence of AAA in RelA wild-type but not in RelA-CKO mice, independent of changes in systolic blood pressure. Furthermore, mesenchymal cell-specific RelA-CKO mice exhibited decreased expression of IL-6 and IL-1β cytokines and decreased recruitment of C68+ and F4/80lo•Ly6Chi monocytes during Ang II infusion. Fibrogenic mesenchymal RelA plays a causal role in Ang II-induced vascular inflammation and AAA in normolipidemic mice.

Assessment of Vascular Dysfunction and Inflammation Induced by Angiotensin II in Mice.

Vascular inflammation in cardiovascular diseases is recognized to be linked with immune cell activation. Recruitment of immune cells into the vessel wall is an early step in angiotensin II-induced vascular dysfunction and arterial hypertension. Exploring the role of monocytes and macrophages in angiotensin II-induced hypertension and vascular inflammation in mouse models highlights the importance of these pathophysiological processes. Here we describe our routinely used protocols concerning angiotensin II-induced hypertension, assessment of blood pressure, vascular function, and immune cell infiltration.

Renal denervation attenuates aldosterone expression and associated cardiovascular pathophysiology in angiotensin II-induced hypertension.

The sympathetic nervous system interacts with the renin-angiotensin-aldosterone system (RAAS) contributing to cardiovascular diseases. In this study, we sought to determine if renal denervation (RDN) inhibits aldosterone expression and associated cardiovascular pathophysiological changes in angiotensin II (Ang II)-induced hypertension. Bilateral RDN or SHAM operation was performed before chronic 14-day Ang II subcutaneous infusion (200ng/kg/min) in male Sprague-Dawley rats. Bilateral RDN blunted Ang II-induced hypertension and ameliorated the mesenteric vascular dysfunction. Cardiovascular hypertrophy in response to Ang II was significantly attenuated by RDN as shown by histopathology and transthoracic echocardiography. Moreover, Ang II-induced vascular and myocardial inflammation and fibrosis were suppressed by RDN with concurrent decrease in fibronectin and collagen deposition, macrophage infiltration, and MCP-1 expression. Interestingly, RDN also inhibited Ang II-induced aldosterone expression in the plasma, kidney and heart. This was associated with the reduction of calcitonin gene-related peptide (CGRP) in the adrenal gland. Ang II promoted aldosterone secretion which was partly attenuated by CGRP in the adrenocortical cell line, suggesting a protective role of CGRP in this model. Activation of transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinases (MAPKs) signaling pathway was both inhibited by RDN especially in the heart. These results suggest that the regulation of the renal sympathetic nerve in Ang II-induced hypertension and associated cardiovascular pathophysiological changes is likely mediated by aldosterone, with CGRP involvement.

Abstract 11888: MyD88 Expressed by Myeloid Cells is Essential for Angiotensin II-Induced Vascular Dysfunction, Inflammation and Arterial Hypertension

Background: Angiotensin II (ATII) causes hypertension and promotes infiltration of inflammatory cells into the vessel wall. Vascular superoxide formation and shear stress mediated inflammatory remodeling of conduit arteries was shown to be myeloid differentiation factor 88 (MyD88) dependent, but the exact mechanism are unknown. Objective: The goal of this study was to determine the mechanism, how MyD88 contributes to the development of ATII-induced vascular dysfunction and arterial hypertension. Methods and Results: MyD88 deficiency profoundly attenuated ATII-induced (1 mg/kg/d for 7 days) blood pressure increase (measured by radio telemetry) and vascular dysfunction (assessed by aortic ring relaxation studies). Additionally vascular superoxide levels as well as mRNA expression levels of VCAM-1, iNOS, Nox2 were decreased in ATII-infused MyD88 -/- mice compared to WT controls. Aortic flow cytometric analysis revealed that ATII-induced infiltration with CD11b + Ly6C high inflammatory monocytes was significantly dampened in MyD88 -/- mice. ATII led to an increased expression of inflammatory monocyte markers in blood and aorta, which was blocked in MyD88 -/- mice, indicating a role of MyD88 for myeloid cell differentiation. Additionally less IFN-gamma + NK cells were detected in the vessel wall of ATII-treated MyD88 -/- mice and aortic lysates showed reduced mRNA levels of several proinflammatory cytokines like IL-12 and IL-1beta. Bone marrow transfer experiments further revealed a protective effect of MyD88 deficiency in inflammatory cells represented by a reduction of vascular dysfunction and inflammation. Conclusion: We provide first evidence that MyD88 expressed by bone marrow-derived cells plays an essential role in ATII-induced vascular dysfunction and arterial hypertension. Our data indicate that MyD88 is important for cytokine production and might be required for ATII-induced differentiation of monocytes into an inflammatory phenotype.

Adiponectin attenuates abdominal aortic aneurysm formation in hyperlipidemic mice

ObjectiveAbdominal aortic aneurysms (AAA) are age-associated, life-threatening inflammatory dilations of the abdominal aorta. Human population studies have shown an association between obesity and AAA formation, but the molecular mechanisms underlying this connection remain largely unexplored. Adiponectin is an anti-inflammatory adipokine that is downregulated in obesity. In this study we evaluated the role of adiponectin in a model of AAA using apolipoprotein E/adiponectin double-knockout (Apoe−/−Apn−/−) mice. Approach and resultsAngiotensin II (Ang II)-infusion in male Apoe−/−Apn−/− mice led to a higher incidence of AAA and a significant increase of maximal aortic diameter compared with that of Apoe−/− mice (2.12 ± 0.07 mm vs. 1.67 ± 0.09 mm, respectively at 28 days). Adiponectin deficiency augmented the early infiltration of macrophages and increased the expression of pro-inflammatory factors in the dilated aortic wall. MMP-2 and MMP-9 activation was also augmented in the aorta of Apoe−/−Apn−/− mice compared to Apoe−/− mice. These data suggest that the downregulation of adiponectin could directly contribute to the elevated incidence of AAA observed in obese individuals. ConclusionsAdiponectin attenuates Ang II-induced vascular inflammation and AAA formation in mice.

Abstract 290: Noncanonical Wnt Signaling Contributes to Angiotensin II-Induced Vascular Inflammation and Abdominal Aortic Aneurysm Formation

Background: Abdominal aortic aneurysms (AAA) are life-threatening localized dilations of the abdominal aorta. A precise knowledge of the molecular mechanisms underlying this disease is required, especially because there are no validated drugs for its treatment. Wnt proteins are secreted signaling molecules that play fundamental roles during embryo development and in the adult. There are 19 Wnt family members in mammals, which frequently have overlapping or redundant functions. Wnt5a is a particularly unique Wnt protein that induces non-canonical Wnt signaling and appears to modulate innate immunity and several signaling pathways involved in vascular inflammation. However, its role in the development of macrovascular pathologies remains unexplored. HYPOTHESIS. Wnt5a-mediated non-canonical Wnt signaling contributes to vascular inflammation and AAA development. METHODS: Cre-loxP strategies were used to generate whole-body Wnt5a-deficient mice, myeloid- Wnt5a-deficient mice and myeloid Wnt5a-overexpressing mice. AAAs were induced in these mouse strains in an apolipoprotein E deficient (apoE-KO) or wild-type (apoE-WT) background by Angiotensin II (Ang II) infusion. Ultrasonography was used to evaluate AAA progression. AAA samples were harvested for histology and gene expression analysis. RESULTS. Whole-body ablation of Wnt5a inhibited Ang II-induced vascular inflammation and AAA formation in apoE-KO mice. Myeloid-specific Wnt5a ablation reduced AAA size, incidence rate and severity, suggesting that myeloid cells are a significant source of pathogenic Wnt5a. Conversely, myeloid-restricted Wnt5a-overexpression led to rapid formation and rupture of dissecting AAAs in Ang II-infused apoE-KO and apoE-WT mice. In human AAAs, Wnt5a expression is significantly increased both at the transcript and protein levels. Mechanistically, Wnt5a appears to promote AAA formation at least in part by promoting vascular inflammation and extracellular matrix remodeling via augmented recruitment of immune cells to the vessel wall. CONCLUSION: Wnt5a-mediated non-canonical Wnt signaling plays a major role in inflammatory AAA formation and may be a potential target for novel AAA therapies.

Abstract 300: Targeted Deletion of Matrix Metalloproteinase 2 Prevents Angiotensin II-Induced Endothelial Dysfunction and Vascular Remodeling, Oxidative Stress and Inflammation

Background: Matrix metalloproteinase 2 (MMP2) is involved in vascular remodeling in atherosclerosis. Whether MMP2 plays a role in angiotensin (Ang) II-induced hypertension, vascular remodeling, oxidative stress and inflammation is unknown. We hypothesized that Mmp2 knockout will prevent Ang II-induced vascular injury. Methods: Ten to 12-week-old male Mmp2 knockout ( Mmp2 -/- ) and wild type (WT) mice were infused with Ang II (1000 ng/kg/min, sc) for 14 days. Systolic blood pressure (SBP) was measured by telemetry. Mesenteric arteries (MA) were studied by pressurized myography. NADPH oxidase activity was evaluated by lucigenin chemiluminescence, and aortic reactive oxygen species (ROS) generation using dihydroethidium staining. Aortic expression of vascular cell adhesion protein 1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), and monocyte/macrophage infiltration were assessed by immunofluorescence. Spleen T cells and monocytes were assessed by flow cytometry. Results: Ang II increased SBP by 50 mmHg ( P <0.01), decreased vasodilatory responses to acetylcholine by 70 % ( P <0.01), induced MA hypertrophic remodeling, indicated by a 1.5-fold increase ( P <0.01) in media-to-lumen ratio and 1.3-fold increase ( P <0.05) in media cross-sectional area, and enhanced MA stiffness ( P <0.01), as shown by a leftward shift of the stress/strain relationship, in WT mice. Ang II increased NADPH oxidase activity 1.4-fold in aorta ( P <0.05), 2-fold in the heart ( P <0.01) and 2.6-fold in the renal cortex ( P <0.01) and aortic ROS generation 25-fold in WT mice ( P <0.01). Ang II increased aortic VCAM-1 and MCP-1 expression 3- and 6-fold, respectively, and monocyte/macrophage infiltration 8-fold in WT ( P <0.05). Ang II increased ≥1.8-fold spleen activated CD4 + CD69 + and CD8 + CD69 + T cells and pro-inflammatory Ly-6C hi monocytes ( P <0.001) in WT mice. Mmp knockout prevented or reduced all of the above except SBP elevation ( P <0.05). Conclusion: MMP2 plays a role in Ang II-induced endothelial dysfunction, vascular remodeling, oxidative stress and inflammation but not in blood pressure elevation.