Basement Membranes and Associated Protein Factors In Development and Disease

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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. Perlecan regulates pericyte dynamics in repair of the blood-brain barrier after ischemic stroke: We have been studying the role of perlecan, a major heparan sulfate proteoglycan of all basement membranes and cartilage, in development and diseases. Blood-brain barrier (BBB) breakdown occurs when the integrity of BBB components is lost as a consequence of ischemic stroke. Perlecan is expressed by endothelial cells (ECs) and is adjacent to pericytes (PCs), suggesting supportive functions in the BBB. Recent studies highlight the importance of PCs in the process of repairing BBB functions, which is triggered by the upregulation of platelet-derived growth factor receptor (PDGFR). We hypothesized that perlecan may play a protective role in BBB maintenance through interaction with pericytes during the repair of the BBB disruption. We induced a 60-minute middle cerebral artery occlusion (MCAO) in adult conditional perlecan-deficient (Perlecan-/--Tg) mice, which express the perlecan transgene only in cartilage to rescue the perinatal lethality of Perlecan-/- mice. Recombinant perlecan C-terminal domain V (DV) was used for in vitro assays. Perlecan-/--Tg mice exhibited larger infarct volumes and more BBB leakage than the control mice on post-surgery day (PSD) 2 after MCAO. While the control mice showed increased numbers of PDGFR-positive PCs around the ischemic lesion on PSD 3, this upregulation was inhibited in Perlecan-/--Tg mice, suggesting that perlecan may participate in PC activation to repair BBB breakdown. In wild-type mice, the expression of integrin alpha5, the subunit of a potential receptor for perlecan, was upregulated both in PCs and in ECs in the ischemic lesion. Using cultured PCs and perlecan DV, we found that PCs attached to DV through integrin alpha5-beta1 and that DV enhanced the PDGF-BB-induced phosphorylation of PDGFR, in addition to that of SHP-2 and FAK, which are downstream molecules of both PDGFR and integrin singnaling. Moreover, DV induced well organized actin stress fibers and focal adhesions in PCs and augmented the PC migration induced by PDGF-BB. These results revealed that perlecan regulates the activation of PCs through the cooperative function of PDGFR and integrin alpha5-beta1, contributing to repair of the BBB after ischemic stroke. Perlecan DV may provide a potential therapeutic for repair of the disrupted BBB in ischemic stroke. Fibulin-7 and its C-terminal fragment have anti-angiogenesis activity 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, 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 pro-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 to compare the levels of neovascularization with 7KCh alone implants. Our results suggest that both Fbln7-FL and Fbln7-C show inhibition of neovascularization in corneas. Fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo: Fbln7 is also expressed in immune-privileged tissues such as eye and placenta, but its functional significance is largely unknown. Using human monocytes and a lipopolysaccharide (LPS)-induced systemic inflammation mouse model, we showed that recombinant full-length Fbln7 (Fbln7-FL) and Fbln7-C proteins modulate monocyte functions. Our data showed that human monocytes adhere to both full-length Fbln7 (Fbln7-FL) and Fbln7-C partially via integrins alpha5-beta1 and alpha2-beta1. Although both Fbln7-FL and Fbln7-C inhibited the activation and differentiation of human monocytes, Fbln7-C had significantly stronger negative effects on cell spreading and stress fiber formation, and on the production of inflammatory mediators as compared to Fbln7-FL. Furthermore, in endotoxemic animals, Fbln7-C, and to a moderate extent Fbln7-FL, reduced the infiltration of macrophages into an inflamed peritoneum. Thus, these results suggest that Fbln7 and its C-terminal fragment could modulate the activity of immune cells and could have therapeutic potential for inflammatory diseases.