Kunio Yasunaga

Angiopoietin-related growth factor antagonizes obesity and insulin resistance

Angiopoietin-related growth factor (AGF), a member of the angiopoietin-like protein (Angptl) family, is secreted predominantly from the liver into the systemic circulation. Here, we show that most (>80%) of the AGF-deficient mice die at about embryonic day 13, whereas the surviving AGF-deficient mice develop marked obesity, lipid accumulation in skeletal muscle and liver, and insulin resistance accompanied by reduced energy expenditure relative to controls. In parallel, mice with targeted activation of AGF show leanness and increased insulin sensitivity resulting from increased energy expenditure. They are also protected from high-fat diet–induced obesity, insulin resistance and nonadipose tissue steatosis. Hepatic overexpression of AGF by adenoviral transduction, which leads to an approximately 2.5-fold increase in serum AGF concentrations, results in a significant (P < 0.01) body weight loss and increases insulin sensitivity in mice fed a high-fat diet. This study establishes AGF as a new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance.

Angiopoietin-related/angiopoietin-like proteins regulate angiogenesis

A general understanding of the molecular mechanisms underlying angiogenesis is emerging from the analysis of targeted mutations in vasculature-related genes.These analyses reveal that angiopoietin signaling through the TIE2 receptor is involved in regulating angiogenesis. Recently, we and several other groups have independently identified several molecules containing a coiled-coil domain and a fibrinogen-like domain, both of which are structurally conserved in angiopoietins. Because these molecules do not bind to the angiopoietin-specific receptor,TIE2, they have been named angiopoietin-related proteins (ARPs) or angiopoietin-like proteins (Angptls).ARPs/Angptls, which are all currently orphan ligands, also have potent activity for regulating angiogenesis as proangiogenic or antiangiogenic factors, suggesting that their receptors may be expressed on endothelial cells. In addition, ARPs/Angptls show pleiotropic effects not only on vascular cells but also on cells of other lineages, such as skin and chondrocyte cells. More recent studies have proposed that ARPs/Angptls are involved in various pathologies, such as tumor angiogenesis and metabolic diseases. To summarize the current findings relating to these proteins, we focus in this review on the functions of ARPs/Angptls as new angiogenic modulating factors in the vascular system and discuss the pleiotropic functions of ARPs/Angptls in nonvascular cell lineages.

The Fibronectin Extra Domain A Activates Matrix Metalloproteinase Gene Expression by an Interleukin-1-dependent Mechanism

The extra domain-A (EDA), present in fibronectin (FN) molecules arising from alternatively spliced transcripts, appears only during specific biological and pathogenic processes. However, its function is poorly understood. To define the physiologic role of this domain in joint connective tissue, the biological effects on rabbit cartilage explants, chondrocytes, and synovial cells were studied. A recombinant EDA protein (rEDA) increased proteoglycan release (3.6-fold) in cartilage explant cultures and markedly induced production of matrix metalloproteinase (MMP)-1 in chondrocytes. In addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These effects were elicited only by rEDA, while its neighboring type III repeats, III11 or III12, scarcely had any such effects. Interestingly, reorganization of F-actin stress fibers accompanied MMP-1 expression in synovial cells treated with rEDA, suggesting alteration of cellular phenotype. Subsequent Northern blotting revealed expression of pro-inflammatory cytokines, including interleukin (IL)-1α and IL-1β, was induced by rEDA prior to MMP-1 expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s promote MMP-1 expression in an autocrine manner. This hypothesis is supported by the reduction of EDA-induced MMP-1 production by IL-1 receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the NH2 or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH2 or COOH terminus of EDA domain by proteolytic cleavage releases the inducing activity. In agreement with these results, full-length cellular FN did not induce MMP-1 production. Furthermore, a 160-kDa EDA-positive FN fragment, which was purified from human placental tissue and corresponds to the region from NH2terminus through the EDA, induced MMP-1 production. Taken together, these results suggest that the EDA in FN fragments triggers alterations of cell physiology and plays a role in matrix degradation in joint connective tissue.

Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration.

We report here the identification of an angiopoietin-related growth factor (AGF). To examine the biological function of AGF in vivo, we created transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF mice exhibited swollen and reddish ears, nose and eyelids. Histological analyses of K14-AGF mice revealed significantly thickened epidermis and a marked increase in proliferating epidermal cells as well as vascular cells in the skin compared with nontransgenic controls. In addition, we found rapid wound closure in the healing process and an unusual closure of holes punched in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in platelets and mast cells, and detected at wounded skin, whereas there was no expression of AGF detected in normal skin tissues, suggesting that AGF derived from these infiltrated cells affects epidermal proliferation and thereby plays a role in the wound healing process. These findings demonstrate that biological functions of AGF in epidermal keratinocytes could lead to novel therapeutic strategies for wound care and epidermal regenerative medicine.

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.

Effect of the sugar chain of soluble recombinant CD59 on complement inhibitory activity

A soluble recombinant CD59#77 (rCD59#77), consisting of 77 amino acids starting from the N terminus of membrane‐bound CD59, was prepared using a gene expression system in CHO cells. The rCD59#77 preparation was composed of glycosylated and non‐glycosylated forms (G and NG forms). Unexpectedly, NG form was 7 times more potent than G form in complement inhibitory activity. Postulating that sialic acids on G‐form molecules make it difficult for rCD59#77 to access nascent membrane attack complexes on the cell surface, the sialic acids were removed by neuraminidase treatment. However, the inhibitory activity was not changed. Next, one of two putative N‐glycosylation sites was mutated by substituting Gln18 for Asn18. The mutant, designated rCD59#77(N/Q), had no sugar moiety and was as active as the NG form of rCD59#77. These results suggest that the bulky sugar moiety at Asn18 is not necessary for the complement‐inhibitory activity of rCD59 and actually hampers that function.