Basement membrane-like structures containing NTH α1(IV) are formed around the endothelial cell network in a novel in vitro angiogenesis model

Angiogenesis is a process through which new blood vessels are formed by sprouting and elongating from existing blood vessels. Several methods have been used to replicate angiogenesis in vitro, including culturing vascular endothelial cells on Matrigel and coculturing with endothelial cells and fibroblasts. However, the angiogenesis elongation process has not been completely clarified in these models. We therefore propose a new in vitro model of angiogenesis, suitable for observing vascular elongation, by seeding a spheroid cocultured from endothelial cells and fibroblasts into a culture dish. In this model, endothelial cells formed tubular networks elongated from the spheroid with a lumen structure and were connected with tight junctions. A basement membrane (BM)-like structure was observed around the tubular network, similarly to blood vessels in vivo. These results suggested that blood vessel-like structure could be reconstituted in our model. Laminin and type IV collagen, main BM components, were highly localized around the network, along with nontriple helical form of type IV collagen α1-chain [NTH α1(IV)]. In an ascorbic acid-depleted condition, laminin and NTH α1(IV) were observed around the network but not the triple-helical form of type IV collagen and the network was unstable. These results suggest that laminin and NTH α1(IV) are involved in the formation of tubular network and type IV collagen is necessary to stabilize the network

Non-Triple Helical Form of Type IV Collagen α1 Chain

Type IV collagen with a triple-helical structure composed of three α chains is a major component of basement membrane. Previously, we reported that non-triple helical form of type IV collagen α1 chain (NTHα1(IV)) was isolated from human placenta and the culture media of human cells. In the present study, we report on the localization of NTH α1(IV) with a monoclonal antibody #370, exclusively reactive for the nascent chain, in the rabbit tissues. The staining was found on the basement membrane of blood vessels, of endomysium, of nerve, and of kidney but not on epithelial basement membrane. In a rabbit angiogenic model, #370 antibody staining was exclusively observed in neovascular tip region of endothelial cells, where no staining with anti-type IV collagen antibody was seen. Distinct localizations suggest that NTHα1(IV) is produced and stably deposited in endothelial cells and the surroundings under physiological conditions with some physiological roles in relation to the dynamics of vascular system

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen