Hiroki Aoki

Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase.

Abdominal aortic aneurysm (AAA) is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is much awaited, few options are available because its molecular pathogenesis remains elusive. Here, we identify JNK as a proximal signaling molecule in the pathogenesis of AAA. Human AAA tissue showed a high level of phosphorylated JNK. We show that JNK programs a gene expression pattern in different cell types that cooperatively enhances the degradation of the extracellular matrix while suppressing biosynthetic enzymes of the extracellular matrix. Selective inhibition of JNK in vivo not only prevented the development of AAA but also caused regression of established AAA in two mouse models. Thus, JNK promotes abnormal extracellular matrix metabolism in the tissue of AAA and may represent a therapeutic target.

Inhibition of protein phosphatase 1 by inhibitor-2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy

The type 1 protein phosphatase (PP1) has been reported to be overactivated in the failing heart, leading to a depression in cardiac function. We investigated whether in vivo PP1 inhibition by myocardial gene transfer of inhibitor‐2 (INH‐2), an endogenous PP1 inhibitor, alleviates heart failure (HF) progression in the cardiomyopathic (CM) hamster, a well‐established HF model. Adenoviral INH‐2 gene delivery improved % fractional shortening of the left ventricle (LV) accompanied by reduced chamber size at 1 wk. In vivo myocardial INH‐2 gene delivery induced an increase in cytosolic PP1 catalytic subunit α (PP1Cα) without inducing the corresponding increase in cytoso‐lic PP1 activity. On the other hand, INH‐2 delivery induced a decrease in microsomal PP1Cα, resulting in a preferential decrease in microsomal PP1 activity, thereby increasing in phospholamban phosphorylation at Ser16. INH‐2 gene transfer alleviated brain natriuretic peptide expression, presumably reflecting improved cardiac function. Moreover, adeno‐associated virus‐mediated INH‐2 gene delivery significantly extended the survival time for 3 mo. These results indicate that increased PP1 activity is an exacerbating factor during progression of genetic cardiomyopathy and modulation of PP1 activity by INH‐2 provides a potential new treatment for HF without activating protein kinase A signaling in cardiomyocytes.— Yamada, M., Ikeda, Y., Yano, M., Yoshimura, K., Nishino, S., Aoyama, H., Wang, L., Aoki, H., and Matsuzaki, M. Inhibition of protein phosphatase 1 by inhibitor‐2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy. FASEB J. 20, E346‐E356 (2006)

Macrophage-Derived Angiopoietin-Like Protein 2 Accelerates Development of Abdominal Aortic Aneurysm

Objective—Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). Methods and Results—Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl2-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. Conclusion—Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.

Inflammasome Activation by Mitochondrial Oxidative Stress in Macrophages Leads to the Development of Angiotensin II–Induced Aortic Aneurysm

Objective—Abdominal aortic aneurysm (AAA) is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of AAA remains unclear. We previously showed that the inflammasome, which regulates the caspase-1–dependent interleukin-1&bgr; production, mediates the sterile cardiovascular inflammatory responses. Therefore, we hypothesized that the inflammasome is a key mediator of initial inflammation in AAA formation. Approach and Results—Apoptosis-associated speck-like protein containing a caspase recruitment domain is highly expressed in adventitial macrophages in human and murine AAA tissues. Using an established mouse model of AAA induced by continuous infusion of angiotensin II in Apoe–/– mice, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe–/– mice were shown to decrease the incidence, maximal diameter, and severity of AAA along with adventitial fibrosis and inflammatory responses significantly, such as inflammatory cell infiltration and cytokine expression in the vessel wall. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe–/– mice also reduced elastic lamina degradation and metalloproteinase activation in the early phase of AAA formation. Furthermore, angiotensin II stimulated generation of mitochondria-derived reactive oxygen species in the adventitial macrophages, and this mitochondria-derived reactive oxygen species generation was inhibited by NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency. In vitro experiments revealed that angiotensin II stimulated the NLRP3 inflammasome activation and subsequent interleukin-1&bgr; release in macrophages, and this activation was mediated through an angiotensin type I receptor/mitochondria-derived reactive oxygen species–dependent pathway. Conclusions—Our results demonstrate the importance of the NLRP3 inflammasome in the initial inflammatory responses in AAA formation, indicating its potential as a novel therapeutic target for preventing AAA progression.

Dynamics of mitochondrial DNA nucleoids regulated by mitochondrial fission is essential for maintenance of homogeneously active mitochondria during neonatal heart development

ABSTRACT Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.

Regression of Abdominal Aortic Aneurysm by Inhibition of c‐Jun N‐Terminal Kinase in Mice

Abstract:  Abdominal aortic aneurysm (AAA) is a common disease that, when surgical treatment is inapplicable, results in rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is eagerly awaited, the destruction of the aortic walls in AAA has been considered an irreversible process. We found that c‐Jun N‐terminal kinase (JNK) is highly activated in human AAA walls. We also found that JNK activity is essential for the expression of matrix metalloproteinase (MMP)‐9 and, concurrently, suppression of the extracellular matrix (ECM) biosynthesis. We therefore investigated the role of JNK in the pathogenesis of AAA in vivo. We created a mouse AAA model by periaortic application of CaCl2, which was accompanied by activation of JNK and MMPs, and suppression of lysyl oxidase (LOX), which is an essential biosynthetic enzyme for collagen and elastin fibers. Our data indicate that, in addition to MMP activities, suppression of ECM biosynthesis may contribute to the AAA pathogenesis because local LOX gene delivery prevented AAA formation. Treatment of mice with SP600125, a specific JNK inhibitor, completely abrogated the formation of CaCl2‐induced AAA. Furthermore, SP600125 treatment after the establishment of AAA caused a reduction in the aortic diameters with normalized tissue architecture. SP600125 treatment also caused significant regression of angiotensin II‐induced AAA in ApoE‐null mice after its establishment, as demonstrated by serial ultrasonographic studies in live animals. These data demonstrate that JNK dictates the abnormal ECM metabolism in AAA pathogenesis by enhancing tissue degradation and suppressing tissue repair. Therefore, inhibition of JNK may provide a novel therapeutic option for AAA.

Inhibition of eNOS phosphorylation mediates endothelial dysfunction in renal failure: new effect of asymmetric dimethylarginine

Patients with chronic kidney disease have elevated circulating asymmetric dimethylarginine (ADMA). Recent studies have suggested that ADMA impairs endothelial nitric oxide synthase (eNOS) by effects other than competition with the substrate L-arginine. Here, we sought to identify the molecular mechanism by which increased ADMA causes endothelial dysfunction in a chronic kidney disease model. In wild-type mice with remnant kidney disease, blood urea nitrogen, serum creatinine, and ADMA were increased by 2.5-, 2-, and 1.2-fold, respectively, without any change in blood pressure. Nephrectomy reduced endothelium-dependent relaxation and eNOS phosphorylation at Ser1177 in isolated aortic rings. In transgenic mice overexpressing dimethylarginine dimethylaminohydrolase-1, the enzyme that metabolizes ADMA, circulating ADMA was not increased by nephrectomy and was decreased to half that of wild-type mice. These mice did not exhibit the nephrectomy-induced inhibition of both endothelium-dependent relaxation and eNOS phosphorylation. In cultured human endothelial cells, agonist-induced eNOS phosphorylation and nitric oxide production were decreased by ADMA at concentrations less than that of L-arginine in the media. Thus, elevated circulating ADMA may be a cause, not an epiphenomenon, of endothelial dysfunction in chronic kidney disease. This effect may be attributable to inhibition of eNOS phosphorylation.

Role of vascular endothelial growth factor-A in development of abdominal aortic aneurysm

AIMS Increased angiogenesis, chronic inflammation, and extracellular matrix degradation are the major pathological features of abdominal aortic aneurysm (AAA). We sought to elucidate the role of vascular endothelial growth factor (VEGF)-A, a potent angiogenic and proinflammatory factor, in the development of AAA. METHODS AND RESULTS Human AAA samples showed increased VEGF-A expression, neovascularization, and macrophage infiltration compared with normal aortic walls. AAA was induced in mice by periaortic application of CaCl(2). AAA mice were treated with soluble VEGF-A receptor (sFlt)-1 or phosphate-buffered saline and sacrificed 6 weeks after the operation. Treatment with sFlt-1 resulted in reduced aneurysm size, restored wavy structure of the elastic lamellae, reduced Mac-2(+) monocytes/macrophages, CD3(+) T-lymphocytes, and CD31(+) vessels, and attenuated matrix metalloproteinase (MMP)-2 and 9 activity in periaortic tissue of AAA. Increased aortic mRNA expression of monocyte chemotactic protein-1, tumour necrosis factor-α, and intercellular adhesion molecule-1 in AAA was attenuated by sFlt-1 treatment. CONCLUSION VEGF-A was overexpressed in the aortic wall of human and experimental AAA. Treatment with sFlt-1 inhibited AAA development in mice, in association with reduced neoangiogenesis, infiltration of inflammatory cells, MMP activity, and extracellular matrix degradation. These findings suggest a crucial role of VEGF-A in the development of AAA.

Isoform-specific roles of protein phosphatase 1 catalytic subunits in sarcoplasmic reticulum-mediated Ca2+ cycling

AIMS protein phosphatase 1 (PP1) is the major isotype of serine/threonine phosphatase in cardiomyocytes, and its activity has been thought to be important for heart failure progression. The PP1 catalytic subunits consist of three distinct genes, PP1α, PP1β/δ, and PP1γ. To date, the function of each PP1 isoform is not well characterized in cardiomyocytes. We sought to determine the functional contribution of each PP1 isoform to sarcoplasmic reticulum (SR)-mediated Ca(2+) cycling in isolated adult rat cardiomyocytes. METHODS AND RESULTS adenoviral vectors encoding short hairpin RNA for each PP1 isoform were transfected into isolated rat cardiomyocytes, and this was followed by analysis of cell shortening, Ca(2+) transients, and the phosphorylation levels of Ca(2+) regulatory proteins. Physical interactions between each PP1 isoform and SR Ca(2+) regulatory proteins were characterized in isolated cardiomyocytes expressing green fluorescent protein (GFP)-tagged PP1 catalytic subunits, and also in canine junctional and longitudinal SR preparations. Successful PP1 isoform knockdown was achieved for each isoform without affecting the expression of the other isoforms. PP1β knockdown most significantly enhanced the Ca(2+) transient and cell shortening by augmenting phospholamban (PLN) phosphorylation at baseline and with low-dose isoproterenol stimulation (10 nM). Interestingly, PP1β was preferentially associated with sarco-endoplasmic ATPase and PLN in GFP-PP1-transfected cardiomyocytes, as well as in canine longitudinal SR preparations. CONCLUSION these findings indicate that PP1β is the most significant PP1 isoform involved in regulating SR Ca(2+) cycling in rat cardiomyocytes.

Inhibition of EP4 Signaling Attenuates Aortic Aneurysm Formation

Background Aortic aneurysm is a common but life-threatening disease among the elderly, for which no effective medical therapy is currently available. Activation of prostaglandin E2 (PGE2) is known to increase the expression of matrix metalloproteinase (MMP) and the release of inflammatory cytokines, and may thus exacerbate abdominal aortic aneurism (AAA) formation. We hypothesized that selective blocking of PGE2, in particular, EP4 prostanoid receptor signaling, would attenuate the development of AAA. Methods and Findings Immunohistochemical analysis of human AAA tissues demonstrated that EP4 expression was greater in AAA areas than that in non-diseased areas. Interestingly, EP4 expression was proportional to the degree of elastic fiber degradation. In cultured human aortic smooth muscle cells (ASMCs), PGE2 stimulation increased EP4 protein expression (1.4±0.08-fold), and EP4 stimulation with ONO-AE1-329 increased MMP-2 activity and interleukin-6 (IL-6) production (1.4±0.03- and 1.7±0.14-fold, respectively, P<0.05). Accordingly, we examined the effect of EP4 inhibition in an ApoE−/− mouse model of AAA infused with angiotensin II. Oral administration of ONO-AE3-208 (0.01–0.5 mg/kg/day), an EP4 antagonist, for 4 weeks significantly decreased the formation of AAA (45–87% reduction, P<0.05). Similarly, EP4+/−/ApoE−/− mice exhibited significantly less AAA formation than EP4+/+/ApoE−/− mice (76% reduction, P<0.01). AAA formation induced by periaortic CaCl2 application was also reduced in EP4+/− mice compared with wild-type mice (73% reduction, P<0.001). Furthermore, in human AAA tissue organ cultures containing SMCs and macrophages, doses of the EP4 antagonist at 10–100 nM decreased MMP-2 activation and IL-6 production (0.6±0.06- and 0.7±0.06-fold, respectively, P<0.05) without increasing MMP-9 activity or MCP-1 secretion. Thus, either pharmacological or genetic EP4 inhibition attenuated AAA formation in multiple mouse and human models by lowering MMP activity and cytokine release. Conclusion An EP4 antagonist that prevents the activation of MMP and thereby inhibits the degradation of aortic elastic fiber may serve as a new strategy for medical treatment of AAA.