Tsuyoshi Kadomatsu

Angiopoietin-like Protein 2 Promotes Chronic Adipose Tissue Inflammation and Obesity-Related Systemic Insulin Resistance

Recent studies of obesity have provided new insights into the mechanisms underlying insulin resistance and metabolic dysregulation. Numerous efforts have been made to identify key regulators of obesity-linked adipose tissue inflammation and insulin resistance. We found that angiopoietin-like protein 2 (Angptl2) was secreted by adipose tissue and that its circulating level was closely related to adiposity, systemic insulin resistance, and inflammation in both mice and humans. Angptl2 activated an inflammatory cascade in endothelial cells via integrin signaling and induced chemotaxis of monocytes/macrophages. Constitutive Angptl2 activation in vivo induced inflammation of the vasculature characterized by abundant attachment of leukocytes to the vessel walls and increased permeability. Angptl2 deletion ameliorated adipose tissue inflammation and systemic insulin resistance in diet-induced obese mice. Conversely, Angptl2 overexpression in adipose tissue caused local inflammation and systemic insulin resistance in nonobese mice. Thus, Angptl2 is a key adipocyte-derived inflammatory mediator that links obesity to systemic insulin resistance.

SIRT7 Controls Hepatic Lipid Metabolism by Regulating the Ubiquitin-Proteasome Pathway

Sirtuins (SIRT1-7) have attracted considerable attention as regulators of metabolism over the past decade. However, the physiological functions and molecular mechanisms of SIRT7 are poorly understood. Here we demonstrate that Sirt7 knockout mice were resistant to high-fat diet-induced fatty liver, obesity, and glucose intolerance, and that hepatic triglyceride accumulation was also attenuated in liver-specific Sirt7 knockout mice. Hepatic SIRT7 positively regulated the protein level of TR4/TAK1, a nuclear receptor involved in lipid metabolism, and as a consequence activated TR4 target genes to increase fatty acid uptake and triglyceride synthesis/storage. Biochemical studies revealed that the DDB1-CUL4-associated factor 1 (DCAF1)/damage-specific DNA binding protein 1 (DDB1)/cullin 4B (CUL4B) E3 ubiquitin ligase complex interacted with TR4, leading to its degradation, while binding of SIRT7 to the DCAF1/DDB1/CUL4B complex inhibited the degradation of TR4. In conclusion, we propose that hepatic SIRT7 controls lipid metabolism in liver by regulating the ubiquitin-proteasome pathway.

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.

Role of Endothelial Cell–Derived Angptl2 in Vascular Inflammation Leading to Endothelial Dysfunction and Atherosclerosis Progression

Objective—Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. Approach and Results—Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E–deficient mice (ApoE−/−/Angptl2−/−) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE−/− mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE−/−/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell–derived nitric oxide. Conversely, Angptl2−/− mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-&kgr;B signaling in endothelial cells and increased monocyte/macrophage chemotaxis. Conclusions—Endothelial cell–derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.

A type I DnaJ homolog, DjA1, regulates androgen receptor signaling and spermatogenesis

Two type I DnaJ homologs DjA1 (DNAJA1; dj2, HSDJ/hdj‐2, rdj1) and DjA2 (DNAJA2; dj3, rdj2) work similarly as a cochaperone of Hsp70s in protein folding and mitochondrial protein import in vitro. To study the in vivo role of DjA1, we generated DjA1‐mutant mice. Surprisingly, loss of DjA1 in mice led to severe defects in spermatogenesis that involve aberrant androgen signaling. Transplantation experiments with green fluorescent protein‐labeled spermatogonia into DjA1−/− mice revealed a primary defect of Sertoli cells in maintaining spermiogenesis at steps 8 and 9. In Sertoli cells of DjA1−/− mice, the androgen receptor markedly accumulated with enhanced transcription of several androgen‐responsive genes, including Pem and testin. Disruption of Sertoli–germ cell adherens junctions was also evident in DjA1−/− mice. Experiments with DjA1−/− fibroblasts and primary Sertoli cells indicated aberrant androgen receptor signaling. These results revealed a critical role of DjA1 in spermiogenesis and suggest that DjA1 and DjA2 are not functionally equivalent in vivo.

Perivascular adipose tissue-secreted angiopoietin-like protein 2 (Angptl2) accelerates neointimal hyperplasia after endovascular injury

Much attention is currently focused on the role of perivascular adipose tissue in development of cardiovascular disease (CVD). Some researchers view it as promoting CVD through secretion of cytokines and growth factors called adipokines, while recent reports reveal that perivascular adipose tissue can exert a protective effect on CVD development. Furthermore, adiponectin, an anti-inflammatory adipokine, reportedly suppresses neointimal hyperplasia after endovascular injury, whereas such vascular remodeling is enhanced by pro-inflammatory adipokines secreted by perivascular adipose, such as tumor necrosis factor-α (TNF-α). These findings suggest that extent of vascular remodeling, a pathological process associated with CVD development, depends on the balance between pro- and anti-inflammatory adipokines secreted from perivascular adipose tissue. We previously demonstrated that angiopoietin-like protein 2 (Angptl2), a pro-inflammatory factor secreted by adipose tissue, promotes adipose tissue inflammation and subsequent systemic insulin resistance in obesity. Here, we examined whether Angptl2 secreted by perivascular adipose tissue contributes to vascular remodeling after endovascular injury in studies of transgenic mice expressing Angptl2 in adipose tissue (aP2-Angptl2 transgenic mice) and Angptl2 knockout mice (Angptl2(-/-) mice). To assess the role of Angptl2 secreted by perivascular adipose tissue on vascular remodeling after endovascular injury, we performed adipose tissue transplantation experiments using these mice. Wild-type mice with perivascular adipose tissue derived from aP2-Angptl2 mice exhibited accelerated neointimal hyperplasia after endovascular injury compared to wild-type mice transplanted with wild-type tissue. Conversely, vascular inflammation and neointimal hyperplasia after endovascular injury were significantly attenuated in wild-type mice transplanted with Angptl2(-/-) mouse-derived perivascular adipose tissue compared to wild-type mice transplanted with wild-type tissue. RT-PCR analysis revealed that mouse Angptl2 expression in perivascular adipose tissue was significantly increased by aging, hypercholesterolemia, and endovascular injury, all risk factors for coronary heart disease (CHD). Immunohistochemical and RT-PCR analysis of tissues from patients with CHD and from non-CHD patients indicated that ANGPTL2 expression in epicardial adipose tissue was unchanged. Interestingly, that analysis also revealed a positive correlation in ANGPTL2 and ADIPONECTIN expression in epicardial adipose tissue of non-CHD patients, a correlation not seen in CHD patients. However, in epicardial adipose tissue from CHD patients, ANGPTL2 expression was positively correlated with that of TNF-α, a correlation was not seen in non-CHD patients. These findings suggest that pro-inflammatory adipokines cooperatively accelerate CHD development and that maintaining a balance between pro- and anti-inflammatory adipokines likely protects non-CHD patients from developing CHD. Overall, our studies demonstrate that perivascular adipose tissue-secreted Angptl2 accelerates vascular inflammation and the subsequent CVD development.

The Secreted Protein ANGPTL2 Promotes Metastasis of Osteosarcoma Cells Through Integrin α5β1, p38 MAPK, and Matrix Metalloproteinases

Preventing signaling by ANGPTL2, which is stimulated by the tumor microenvironment, could inhibit metastasis. Microenvironment Drives Osteosarcoma Metastasis The selective pressures of the tumor microenvironment alter the behavior of cancer cells. Odagiri et al. found that the expression of ANGPTL2, encoding the secreted angiopoietin-like protein 2, increased in osteosarcoma cells grown in xenografts in mice or cultured in conditions that mimic the tumor microenvironment. Silencing ANGPTL2 or overexpressing a proteolytically cleaved form decreased matrix metalloproteinase-9 (MMP-9) activity, delayed the onset of metastasis from xenografts, and prolonged survival in mice. The abundance of ANGPTL2 correlated with that of MMP-9 in patient samples, and both inversely correlated with metastasis-free survival in patients. The findings highlight the influence of the tumor microenvironment and implicate ANGPTL2 as a target to hinder metastasis in osteosarcoma. The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation–related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin α5β1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. Expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

Diverse roles of ANGPTL2 in physiology and pathophysiology

Stresses based on aging and lifestyle can cause tissue damage. Repair of damage by tissue remodeling is often meditated by communications between parenchymal and stromal cells via cell-cell contact or humoral factors. However, loss of tissue homeostasis leads to chronic inflammation and pathological tissue remodeling. Angiopoietin-like protein 2 (ANGPTL2) maintains tissue homeostasis by promoting adaptive inflammation and subsequent tissue reconstruction, whereas excess ANGPTL2 activation induced by prolonged stress promotes breakdown of tissue homeostasis due to chronic inflammation and irreversible tissue remodeling, promoting development of various metabolic diseases. Thus, it is important to define how ANGPTL2 signaling is regulated in order to understand mechanisms underlying disease development. Here, we focus on ANGPTL2 function in physiology and pathophysiology.

Angiopoietin-Related Growth Factor Enhances Blood Flow Via Activation of the ERK1/2-eNOS-NO Pathway in a Mouse Hind-Limb Ischemia Model

Objective—Transgenic mice overexpressing angiopoietin-related growth factor (AGF) exhibit enhanced angiogenesis, suggesting that AGF may be a useful drug target in ischemic disease. Our goal was to determine whether AGF enhances blood flow in a mouse hind-limb ischemia model and to define molecular mechanisms underlying AGF signaling in endothelial cells. Methods and Results—Intramuscular injection of adenovirus harboring AGF into the ischemic limb increased AGF production, which increased blood flow through induction of angiogenesis and arteriogenesis, thereby reducing the necessity for limb amputation. In vitro analysis showed that exposing human umbilical venous endothelial cells to AGF increased nitric oxide (NO) production through activation of an ERK1/2-endothelial NO synthetase (eNOS) signaling pathway. AGF-stimulated eNOS phosphorylation, NO production, and endothelial cell migration were all abolished by specific MEK1/2 inhibitors. Moreover, AGF did not restore blood flow to ischemic hind-limbs of either mice receiving NOS inhibitor L-NAME or eNOS knockout mice. Conclusion—Activation of an ERK1/2-eNOS-NO pathway is a crucial signaling mechanism by which AGF increases blood flow through induction of angiogenesis and arteriogenesis. Further investigation of the regulation underlying AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.

Angiopoietin-like protein 2 accelerates carcinogenesis by activating chronic inflammation and oxidative stress

Chronic inflammation has received much attention as a risk factor for carcinogenesis. We recently reported that Angiopoietin-like protein 2 (Angptl2) facilitates inflammatory carcinogenesis and metastasis in a chemically induced squamous cell carcinoma (SCC) of the skin mouse model. In particular, we demonstrated that Angptl2-induced inflammation enhanced susceptibility of skin tissues to “preneoplastic change” and “malignant conversion” in SCC development; however, mechanisms underlying this activity remain unclear. Using this model, we now report that transgenic mice overexpressing Angptl2 in skin epithelial cells (K14-Angptl2 Tg mice) show enhanced oxidative stress in these tissues. Conversely, in the context of this model, Angptl2 knockout (KO) mice show significantly decreased oxidative stress in skin tissue as well as a lower incidence of SCC compared with wild-type mice. In the chemically induced SCC model, treatment of K14-Angptl2 Tg mice with the antioxidant N-acetyl cysteine (NAC) significantly reduced oxidative stress in skin tissue and the frequency of SCC development. Interestingly, K14-Angptl2 Tg mice in the model also showed significantly decreased expression of mRNA encoding the DNA mismatch repair enzyme Msh2 compared with wild-type mice and increased methylation of the Msh2 promoter in skin tissues. Msh2 expression in skin tissues of Tg mice was significantly increased by NAC treatment, as was Msh2 promoter demethylation. Overall, this study strongly suggests that the inflammatory mediator Angptl2 accelerates chemically induced carcinogenesis through increased oxidative stress and decreased Msh2 expression in skin tissue. Implications: Angptl2-induced inflammation increases susceptibility to microenvironmental changes, allowing increased oxidative stress and decreased Msh2 expression; therefore, Angptl2 might be a target to develop new strategies to antagonize these activities in premalignant tissue. Mol Cancer Res; 12(2); 239–49. ©2013 AACR.