Basement membranes (BMs) are thin extracellular matrices (ECMs) that separate epithelial and mesenchymal cells and surround other cells, such as endothelial, muscle, and neural cells. BMs consist of a unique set of proteins, such as laminins, perlecan, and collagen IV. They are also associated with other ECM proteins, such as fibulin and fibronectin, in various tissues. BMs are the first ECMs to appear during development and are critical for organ development and tissue repair. They provide the scaffold for cells and cell layers and play essential roles in cell adhesion, migration, proliferation, and differentiation during morphogenesis. We have studied the specific functions of basement membranes and their associated proteins to delineate their structural and functional relationships, to elucidate their regulatory mechanisms, and to describe related protein interactions that occur during development and disease. Anti-angiogenic activity of fibulin-7 and its fragment in vivo: Fibulins are secreted glycoproteins that are associated with basement membranes, elastic fibers, and other matrices. Fibulins mediate cell-to-matrix communication, as well as stabilize the extracellular matrix (ECM). We previously identified fibulin-7 (Fbln7), the newest member of the fibulin family, in developing teeth. We showed that Fbln7 is expressed by odontoblasts and is a cell adhesion molecule for dental mesenchyme cells and odontoblasts. We found that Fbln7 interacts with ECM proteins, such as fibronectin, as well as growth factors. Fbln7 is also expressed in vessel walls, cartilage, skin, the eye, and the placenta. ECM proteins and their fragments play an important role in angiogenesis through either the promotion or suppression of angiogenic and inflammatory processes. We showed that Fbln7 is expressed in avascular and immunotolerant tissues, such as cartilage, the eye, and the placenta. We found that recombinant Fbln7 proteins inhibited angiogenesis by blocking tube formation in human umbilical vein endothelial cells (HUVECs). A C-terminal Fbln7 fragment (Fbln7-C) had the strongest inhibitory effect on HUVEC tube formation and on vessel sprouting in aortic ring assays in vitro. Mutations in Fibulin-5 and -6, which are expressed in the choroidal layer surrounding the retina similar to Fbln7, are associated with age-related macular degeneration (AMD) in humans. AMD degeneration is the leading cause of blindness in patients older than 60 years due to choroidal neovascularization, leading to vision loss. We hypothesized that Fbln7 fragments have anti-angiogenic activity in vivo and could be a potential therapeutic compound for AMD. Therefore, we chose a novel and reliable method to study ocular angiogenesis using a rat corneal model in collaboration with Dr. Ignacio Rodriguez and Dr. Juan Amaral at NEI. The method consists of placing an implant containing a pro-inflammatory and -angiogenic lipid, 7-KetoCholesterol (7KCh), within the anterior chamber of the eye. The 7KCh implant causes an inflammatory response 4 days after implantation and induces neovascularization that peaks at day 7. Our strategy is to place biodegradable implants containing either a combination of 7KCh and Fbln7-FL or 7KCh and Fbln7-C fragments, and compare the levels of neovascularization with 7KCh alone implants. Our results suggest that both Fbln7-FL and Fbln7-C show inhibition of neovascularization in the corneas. Identification of Peptides from the C-terminal domain of Fibn7 active for endothelial cell adhesion and tube formation disruption: The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. We screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro anti-angiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and an anti-integrin beta1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. Fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that these peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle: Perlecan (Hspg2) is a major heparan sulfate proteoglycan in basement membranes. Skeletal muscle continuously adapts in response to a variety of stimuli. Mechanical load causes hypertrophy, while physical inactivity leads to atrophy. Muscle mass is in dynamic equilibrium between protein synthesis and protein degradation. The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. In collaboration with Dr. Eri Arikawa-Hirasawa and her colleagues at Juntendo University Medical School, we studied the association between perlecan and autophagy in the slow-twitch soleus muscle using a lethality-rescued perlecan knockout mouse model (Hspg2-/-; Tg), in which the perlecan transgene is expressed specifically in cartilage but not other tissues, including muscles. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This enhancement was accompanied by a decrease in autophagic substrates, and an increase in LC3II levels. Perlecan deficiency also caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPK. Our findings revealed that perlecan inhibits the autophagic process through activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.
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