Chronic intermittent hypoxia facilitates the development of angiotensin II-induced abdominal aortic aneurysm in male mice.

Obstructive sleep apnea (OSA), characterized by episodes of intermittent hypoxia (IH), is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, whether IH serves as an independent risk factor for AAA development remains to be investigated. Here, we determined the effects of chronic (6 mo) IH on angiotensin (Ang II)-induced AAA development in C57BL/6J male mice and investigated the underlying mechanisms of IH in cultured vascular smooth muscle cells (SMCs). IH increased the susceptibility of mice to develop AAA in response to Ang II infusion by facilitating the augmentation of the abdominal aorta's diameter as assessed by transabdominal ultrasound imaging. Importantly, IH with Ang II augmented aortic elastin degradation and the expression of matrix metalloproteinases (MMPs), mainly MMP8, MMP12, and a disintegrin and metalloproteinase-17 (ADAM17) as measured by histology and immunohistochemistry. Mechanistically, IH increased the activities of MMP2, MMP8, MMP9, MMP12, and ADAM17, while reducing the expression of the MMP regulator reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in cultured SMCs. Aortic samples from human AAA were associated with decreased RECK and increased expression of ADAM17 and MMPs. These data suggest that IH facilitates AAA development when additional stressors are superimposed and that this occurs in association with an increased presence of aortic MMPs and ADAM17, potentially due to IH-induced modulation of RECK expression. These findings support a plausible synergistic link between OSA and AAA and provide a better understanding of the molecular mechanisms underlying the pathogenesis of AAA.NEW & NOTEWORTHY IH facilitates Ang II-induced abdominal aortic diameter expansion and AAA development in C57BL/6J male mice. IH upregulates the expression of specific MMPs such as MMP8, MMP12, and ADAM17. IH directly suppresses RECK expression and increases MMPs activity in SMCs. Human AAA tissues exhibit a downregulation of RECK and an upregulation of ADAM17 and MMPs.

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

Insulin-like growth factor 1 reduces coronary atherosclerosis in pigs with familial hypercholesterolemia.

Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Macrophage-Specific IGF-1 Overexpression Reduces CXCL12 Chemokine Levels and Suppresses Atherosclerotic Burden in Apoe-Deficient Mice.

OBJECTIVE: IGF-1 (insulin-like growth factor 1) exerts pleiotropic effects including promotion of cellular growth, differentiation, survival, and anabolism. We have shown that systemic IGF-1 administration reduced atherosclerosis in Apoe-/- (apolipoprotein E deficient) mice, and this effect was associated with a reduction in lesional macrophages and a decreased number of foam cells in the plaque. Almost all cell types secrete IGF-1, but the effect of macrophage-derived IGF-1 on the pathogenesis of atherosclerosis is poorly understood. We hypothesized that macrophage-derived IGF-1 will reduce atherosclerosis. Approach and Results: We created macrophage-specific IGF-1 overexpressing mice on an Apoe-/- background. Macrophage-specific IGF-1 overexpression reduced plaque macrophages, foam cells, and atherosclerotic burden and promoted features of stable atherosclerotic plaque. Macrophage-specific IGF1 mice had a reduction in monocyte infiltration into plaque, decreased expression of CXCL12 (CXC chemokine ligand 12), and upregulation of ABCA1 (ATP-binding cassette transporter 1), a cholesterol efflux regulator, in atherosclerotic plaque and in peritoneal macrophages. IGF-1 prevented oxidized lipid-induced CXCL12 upregulation and foam cell formation in cultured THP-1 macrophages and increased lipid efflux. We also found an increase in cholesterol efflux in macrophage-specific IGF1-derived peritoneal macrophages. CONCLUSIONS: Macrophage IGF-1 overexpression reduced atherosclerotic burden and increased features of plaque stability, likely via a reduction in CXCL12-mediated monocyte recruitment and an increase in ABCA1-dependent macrophage lipid efflux.

PET Imaging Radiotracers of Chemokine Receptors.

Chemokines and chemokine receptors have been recognized as critical signal components that maintain the physiological functions of various cells, particularly the immune cells. The signals of chemokines/chemokine receptors guide various leukocytes to respond to inflammatory reactions and infectious agents. Many chemokine receptors play supportive roles in the differentiation, proliferation, angiogenesis, and metastasis of diverse tumor cells. In addition, the signaling functions of a few chemokine receptors are associated with cardiac, pulmonary, and brain disorders. Over the years, numerous promising molecules ranging from small molecules to short peptides and antibodies have been developed to study the role of chemokine receptors in healthy states and diseased states. These drug-like candidates are in turn exploited as radiolabeled probes for the imaging of chemokine receptors using noninvasive in vivo imaging, such as positron emission tomography (PET). Recent advances in the development of radiotracers for various chemokine receptors, particularly of CXCR4, CCR2, and CCR5, shed new light on chemokine-related cancer and cardiovascular research and the subsequent drug development. Here, we present the recent progress in PET radiotracer development for imaging of various chemokine receptors.

Malignant Peritoneal Mesothelioma Presenting with Polymyalgia Rheumatica-like Syndrome.

A 30-year-old man was admitted to our hospital because of pain in his proximal extremities. The pain mimicked polymyalgia rheumatica (PMR) and it temporarily improved by a low dose of glucocorticoids, but his symptoms relapsed many times. After six years of glucocorticoid treatment, he developed abdominal pain and ascites, for which he was diagnosed with malignant peritoneal mesothelioma (MPM). His PMR-like symptoms improved with cytoreductive surgery and chemotherapy for MPM. Finally, we diagnosed this PMR-like syndrome to be paraneoplastic syndrome with MPM. Although cases of MPM complicated by PMR-like syndrome are rare, MPM should be taken into account in the differential diagnosis.

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation.

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1-100 µM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

SM22α (Smooth Muscle Protein 22-α) Promoter-Driven IGF1R (Insulin-Like Growth Factor 1 Receptor) Deficiency Promotes Atherosclerosis.

Objective- IGF-1 (insulin-like growth factor 1) is a major autocrine/paracrine growth factor, which promotes cell proliferation, migration, and survival. We have shown previously that IGF-1 reduced atherosclerosis and promoted features of stable atherosclerotic plaque in Apoe-/- mice-an animal model of atherosclerosis. The aim of this study was to assess effects of smooth muscle cell (SMC) IGF-1 signaling on the atherosclerotic plaque. Approach and Results- We generated Apoe-/- mice with IGF1R (IGF-1 receptor) deficiency in SMC and fibroblasts (SM22α [smooth muscle protein 22 α]-CreKI/IGF1R-flox mice). IGF1R was decreased in the aorta and adventitia of SM22α-CreKI/IGF1R-flox mice and also in aortic SMC, embryonic, skin, and lung fibroblasts isolated from SM22α-CreKI/IGF1R-flox mice. IGF1R deficiency downregulated collagen mRNA-binding protein LARP6 (La ribonucleoprotein domain family, member 6) and vascular collagen, and mice exhibited growth retardation. The high-fat diet-fed SM22α-CreKI/IGF1R-flox mice had increased atherosclerotic burden and inflammatory responses. α-SMA (α-smooth muscle actin)-positive plaque cells had reduced proliferation and elevated apoptosis. SMC/fibroblast-targeted decline in IGF-1 signaling decreased atherosclerotic plaque SMC, markedly depleted collagen, reduced plaque fibrous cap, and increased plaque necrotic cores. Aortic SMC isolated from SM22α-CreKI/IGF1R-flox mice had decreased cell proliferation, migration, increased sensitivity to apoptosis, and these effects were associated with disruption of IGF-1-induced Akt signaling. Conclusions- IGF-1 signaling in SMC and in fibroblast is a critical determinant of normal vascular wall development and atheroprotection.

Unusual cause of hoarseness: Arytenoid cartilage dislocation without a traumatic event.

Arytenoid cartilage dislocation is a rare but curable cause of hoarseness and is commonly related to intubation or laryngeal trauma. We present a case of arytenoid cartilage dislocation without a traumatic event in a man who complained of acute hoarseness. An 82-year-old man visited our emergency department complaining of acute hoarseness. He had no history of general anesthesia or laryngeal trauma and had not caught a cold. He showed no abnormal physical findings including the pharynx. Examination using a laryngoscope revealed that the left vocal fold was fixed in the paramedian position. We performed contrast-enhanced computed tomography (CT), but we could not find any cause of the left recurrent laryngeal nerve paralysis. His hoarseness persisted for one month and the initial CT was therefore reassessed. It was found that the left arytenoid cartilage was dislocated forward, and we therefore made a diagnosis of arytenoid cartilage dislocation without a traumatic event. We tried to refer him to an otolaryngologist for surgical treatment, but he declined. Fortunately, his hoarseness gradually improved over a period of two months. Arytenoid cartilage dislocation rarely occurs without a traumatic event. CT is useful for accurate diagnosis of this condition. Since early diagnosis can lead to appropriate surgical treatment and improvement in vocal function, the possibility of arytenoid cartilage dislocation should be considered in patients with acute hoarseness without apparent causes.

Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death.

Atherosclerotic plaque destabilization is the major determinant of most acute coronary events. Smooth muscle cell (SMC) death contributes to plaque destabilization. Here, we describe a novel antiapoptotic mechanism in vascular SMCs that involves interaction of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with apurinic/apyrimidinic endonuclease 1 (Ape1), the major oxidized DNA repair enzyme. GAPDH down-regulation potentiated H2O2-induced DNA damage and SMC apoptosis. Conversely, GAPDH overexpression decreased DNA damage and protected SMCs against apoptosis. Ape1 down-regulation reversed the resistance of GAPDH-overexpressing cells to DNA damage and apoptosis, which indicated that Ape1 is indispensable for GAPDH-dependent protective effects. GAPDH bound Ape1 in the SMC nucleus, and blocking (or oxidation) of GAPDH active site cysteines suppressed GAPDH/Ape1 interaction and potentiated apoptosis. GAPDH up-regulated Ape1 via a transcription factor homeobox protein Hox-A5-dependent mechanism. GAPDH levels were reduced in atherosclerotic plaque SMCs, and this effect correlated with oxidative stress and SMC apoptosis. Thus, we demonstrated that nuclear GAPDH/Ape1 interaction preserved Ape1 activity, reduced DNA damage, and prevented SMC apoptosis. Suppression of SMC apoptosis by maintenance of nuclear GAPDH/Ape1 interactions may be a novel therapy to increase atherosclerotic plaque stability.-Hou, X., Snarski, P., Higashi, Y., Yoshida, T., Jurkevich, A., Delafontaine, P., Sukhanov, S. Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death.

TRAF3IP2 mediates atherosclerotic plaque development and vulnerability in ApoE(-/-) mice.

BACKGROUND AND AIMS: Atherosclerosis is a major cause of heart attack and stroke. Inflammation plays a critical role in the development of atherosclerosis. Since the cytoplasmic adaptor molecule TRAF3IP2 (TRAF3-Interacting Protein 2) plays a causal role in various autoimmune and inflammatory diseases, we hypothesized that TRAF3IP2 mediates atherosclerotic plaque development. METHODS: TRAF3IP2/ApoE double knockout (DKO) mice were generated by crossing TRAF3IP2(-/-) and ApoE(-/-) mice. ApoE(-/-) mice served as controls. Both DKO and control mice were fed a high-fat diet for 12 weeks. Plasma lipids were measured by ELISA, atherosclerosis by en face analysis of aorta and plaque cross-section measurements at the aortic valve region, plaque necrotic core area, collagen and smooth muscle cell (SMC) content by histomorphometry, and aortic gene expression by RT-qPCR. RESULTS: The plasma lipoprotein profile was not altered by TRAF3IP2 gene deletion in ApoE(-/-) mice. While total aortic plaque area was decreased in DKO female, but not male mice, the plaque necrotic area was significantly decreased in DKO mice of both genders. Plaque collagen and SMC contents were increased significantly in both female and male DKO mice compared to respective controls. Aortic expression of proinflammatory cytokine (Tumor necrosis factor α, TNFα), chemokine (Chemokine (C-X-C motif) Ligand 1, CXCL1) and adhesion molecule (Vascular cell adhesion molecule 1, VCAM1; and Intercellular adhesion molecule 1, ICAM1) gene expression were decreased in both male and female DKO mice. In addition, the male DKO mice expressed markedly reduced levels of extracellular matrix (ECM)-related genes, including TIMP1 (Tissue inhibitor of metalloproteinase 1), RECK (Reversion-Inducing-Cysteine-Rich Protein with Kazal Motifs) and ADAM17 (A Disintegrin And Metalloproteinase 17). CONCLUSIONS: TRAF3IP2 plays a causal role in atherosclerotic plaque development and vulnerability, possibly by inducing the expression of multiple proinflammatory mediators. TRAF3IP2 could be a potential therapeutic target in atherosclerotic vascular diseases.

Insulin-Like Growth Factor-1 Receptor Deficiency in Macrophages Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Apolipoprotein E-Deficient Mice.

BACKGROUND: We have previously shown that systemic infusion of insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory and antioxidant effects and reduces atherosclerotic burden in apolipoprotein E (Apoe)-deficient mice. Monocytes/macrophages express high levels of IGF-1 receptor (IGF1R) and play a pivotal role in atherogenesis, but the potential effects of IGF-1 on their function are unknown. METHODS AND RESULTS: To determine mechanisms whereby IGF-1 reduces atherosclerosis and to explore the potential involvement of monocytes/macrophages, we created monocyte/macrophage-specific IGF1R knockout (MΦ-IGF1R-KO) mice on an Apoe(-/-) background. We assessed atherosclerotic burden, plaque features of stability, and monocyte recruitment to atherosclerotic lesions. Phenotypic changes of IGF1R-deficient macrophages were investigated in culture. MΦ-IGF1R-KO significantly increased atherosclerotic lesion formation, as assessed by Oil Red O staining of en face aortas and aortic root cross-sections, and changed plaque composition to a less stable phenotype, characterized by increased macrophage and decreased α-smooth muscle actin-positive cell population, fibrous cap thinning, and decreased collagen content. Brachiocephalic artery lesions of MΦ-IGF1R-KO mice had histological features implying plaque vulnerability. Macrophages isolated from MΦ-IGF1R-KO mice showed enhanced proinflammatory responses on stimulation by interferon-γ and oxidized low-density lipoprotein and elevated antioxidant gene expression levels. Moreover, IGF1R-deficient macrophages had decreased expression of ABCA1 and ABCG1 and reduced lipid efflux. CONCLUSIONS: Our data indicate that macrophage IGF1R signaling suppresses macrophage and foam cell accumulation in lesions and reduces plaque vulnerability, providing a novel mechanism whereby IGF-1 exerts antiatherogenic effects.

Interaction between insulin-like growth factor-1 and atherosclerosis and vascular aging.

The process of vascular aging encompasses alterations in the function of endothelial (ECs) and vascular smooth muscle cells (VSMCs) via oxidation, inflammation, cell senescence and epigenetic modifications, increasing the probability of atherosclerosis. Aged vessels exhibit decreased endothelial antithrombogenic properties, increased reactive oxygen species generation, inflammatory signaling and migration of VSMCs to the subintimal space, impaired angiogenesis and increased elastin degradation. The key initiating step in atherogenesis is subendothelial accumulation of apolipoprotein B-containing low-density lipoproteins resulting in activation of ECs and recruitment of monocytes. Activated ECs secrete 'chemokines' that interact with cognate chemokine receptors on monocytes and promote directional migration. Recruitment of immune cells establishes a proinflammatory status, further causing elevated oxidative stress, which in turn triggers a series of events including apoptotic or necrotic death of vascular and nonvascular cells. Increased oxidative stress is also considered to be a key factor in mechanisms of aging-associated changes in tissue integrity and function. Experimental evidence indicates that insulin-like growth factor-1 exerts antioxidant, anti-inflammatory and pro-survival effects on the vasculature, reducing atherosclerotic plaque burden and promoting features of atherosclerotic plaque stability.

Angiotensin II inhibits satellite cell proliferation and prevents skeletal muscle regeneration.

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Although patients with advanced CHF or CKD often have increased angiotensin II (Ang II) levels and cachexia and Ang II causes skeletal muscle wasting in rodents, the potential effects of Ang II on muscle regeneration are unknown. Muscle regeneration is highly dependent on the ability of a pool of muscle stem cells (satellite cells) to proliferate and to repair damaged myofibers or form new myofibers. Here we show that Ang II reduced skeletal muscle regeneration via inhibition of satellite cell (SC) proliferation. Ang II reduced the number of regenerating myofibers and decreased expression of SC proliferation/differentiation markers (MyoD, myogenin, and active-Notch) after cardiotoxin-induced muscle injury in vivo and in SCs cultured in vitro. Ang II depleted the basal pool of SCs, as detected in Myf5(nLacZ/+) mice and by FACS sorting, and this effect was inhibited by Ang II AT1 receptor (AT1R) blockade and in AT1aR-null mice. AT1R was highly expressed in SCs, and Notch activation abrogated the AT1R-mediated antiproliferative effect of Ang II in cultured SCs. In mice that developed CHF postmyocardial infarction, there was skeletal muscle wasting and reduced SC numbers that were inhibited by AT1R blockade. Ang II inhibition of skeletal muscle regeneration via AT1 receptor-dependent suppression of SC Notch and MyoD signaling and proliferation is likely to play an important role in mechanisms leading to cachexia in chronic disease states such as CHF and CKD.

Molecular mechanisms and signaling pathways of angiotensin II-induced muscle wasting: potential therapeutic targets for cardiac cachexia.

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Many factors are involved in the development of cachexia, and there is increasing evidence that angiotensin II (Ang II), the main effector molecule of the renin-angiotensin system (RAS), plays an important role in this process. Patients with advanced CHF or CKD often have increased Ang II levels and cachexia, and angiotensin-converting enzyme (ACE) inhibitor treatment improves weight loss. In rodent models, an increase in systemic Ang II leads to weight loss through increased protein breakdown, reduced protein synthesis in skeletal muscle and decreased appetite. Ang II activates the ubiquitin-proteasome system via generation of reactive oxygen species and via inhibition of the insulin-like growth factor-1 signaling pathway. Furthermore, Ang II inhibits 5' AMP-activated protein kinase (AMPK) activity and disrupts normal energy balance. Ang II also increases cytokines and circulating hormones such as tumor necrosis factor-α, interleukin-6, serum amyloid-A, glucocorticoids and myostatin, which regulate muscle protein synthesis and degradation. Ang II acts on hypothalamic neurons to regulate orexigenic/anorexigenic neuropeptides, such as neuropeptide-Y, orexin and corticotropin-releasing hormone, leading to reduced appetite. Also, Ang II may regulate skeletal muscle regenerative processes. Several clinical studies have indicated that blockade of Ang II signaling via ACE inhibitors or Ang II type 1 receptor blockers prevents weight loss and improves muscle strength. Thus the RAS is a promising target for the treatment of muscle atrophy in patients with CHF and CKD. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

Angiotensin II infusion induces marked diaphragmatic skeletal muscle atrophy.

Advanced congestive heart failure (CHF) and chronic kidney disease (CKD) are characterized by increased angiotensin II (Ang II) levels and are often accompanied by significant skeletal muscle wasting that negatively impacts mortality and morbidity. Both CHF and CKD patients have respiratory muscle dysfunction, however the potential effects of Ang II on respiratory muscles are unknown. We investigated the effects of Ang II on diaphragm muscle in FVB mice. Ang II induced significant diaphragm muscle wasting (18.7±1.6% decrease in weight at one week) and reduction in fiber cross-sectional area. Expression of the E3 ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) and of the pro-apoptotic factor BAX was increased after 24 h of Ang II infusion (4.4±0.3 fold, 3.1±0.5 fold and 1.6±0.2 fold, respectively, compared to sham infused control) suggesting increased muscle protein degradation and apoptosis. In Ang II infused animals, there was significant regeneration of injured diaphragm muscles at 7 days as indicated by an increase in the number of myofibers with centralized nuclei and high expression of embryonic myosin heavy chain (E-MyHC, 11.2±3.3 fold increase) and of the satellite cell marker M-cadherin (59.2±22.2% increase). Furthermore, there was an increase in expression of insulin-like growth factor-1 (IGF-1, 1.8±0.3 fold increase) in Ang II infused diaphragm, suggesting the involvement of IGF-1 in diaphragm muscle regeneration. Bone-marrow transplantation experiments indicated that although there was recruitment of bone-marrow derived cells to the injured diaphragm in Ang II infused mice (267.0±74.6% increase), those cells did not express markers of muscle stem cells or regenerating myofibers. In conclusion, Ang II causes marked diaphragm muscle wasting, which may be important for the pathophysiology of respiratory muscle dysfunction and cachexia in conditions such as CHF and CKD.

Angiotensin II upregulates protein phosphatase 2Cα and inhibits AMP-activated protein kinase signaling and energy balance leading to skeletal muscle wasting.

Congestive heart failure and chronic kidney disease are characterized by chronically elevated angiotensin II (Ang II) and muscle wasting. Ang II causes skeletal muscle wasting by reducing appetite and by enhancing catabolism. The serine/threonine kinase AMP-activated protein kinase (AMPK) functions mainly as a sensor of cellular energy status. It is energy sparing and favors ATP generation. We hypothesized that Ang II induces muscle wasting in part by inhibiting AMPK signaling and altering cellular energy balance. Our results show that Ang II infusion in mice reduced gastrocnemius muscle weight by 26% and depleted ATP by 74%. In addition, Ang II upregulated protein phosphatase 2Cα by 2.6-fold and reduced AMPK phosphorylation and signaling in muscle. Importantly, the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside restored AMPK activity to levels of pair-fed controls and reversed Ang II-mediated ATP depletion and muscle wasting. Moreover, 5-aminoimidazole-4-carboxamide ribonucleoside activated Akt and inhibited Ang II-induced increases in E3 ubiquitin ligase expression. These novel results demonstrate critical roles for energy depletion and AMPK inhibition in Ang II-induced skeletal muscle wasting and suggest a therapeutic potential for AMPK activators in diseases characterized by muscle wasting.

Differential requirement for nitric oxide in IGF-1-induced anti-apoptotic, anti-oxidant and anti-atherosclerotic effects.

We have shown previously that insulin like-growth factor I (IGF-1) suppressed atherosclerosis in Apoe(-/-) mice and activated endothelial nitric oxide (NO) synthase. To determine whether IGF-1-induced atheroprotection depends on NO, IGF-1- or saline-infused mice were treated with l-NAME, the pan-NO synthase inhibitor or with d-NAME (control). IGF-1 reduced atherosclerosis in both the d-NAME and l-NAME groups suggesting that IGF-1's anti-atherogenic effect was NO-independent. IGF-1 increased plaque smooth muscle cells, suppressed cell apoptosis and downregulated lipoprotein lipase and these effects were also NO-independent. On the contrary, IGF-1 decreased oxidative stress and suppressed TNF-α levels and these effects were blocked by l-NAME. Thus IGF-1's anti-oxidant effect is dependent on its ability to increase NO but is distinct from its anti-atherosclerotic effect which is NO-independent.