Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition.

Collagen XVIII is an evolutionary conserved ubiquitously expressed basement membrane proteoglycan produced in three isoforms via two promoters (P). Here, we assess the function of the N-terminal, domain of unknown function/frizzled-like sequences unique to medium/long collagen XVIII by creating P-specific null mice. P2-null mice, which only produce short collagen XVIII, developed reduced bulk-adiposity, hepatic steatosis, and hypertriglyceridemia. These abnormalities did not develop in P1-null mice, which produce medium/long collagen XVIII. White adipose tissue samples from P2-null mice contain larger reserves of a cell population enriched in early adipocyte progenitors; however, their embryonic fibroblasts had ∼ 50% lower adipocyte differentiation potential. Differentiating 3T3-L1 fibroblasts into mature adipocytes produced striking increases in P2 gene-products and dramatic falls in P1-transcribed mRNA, whereas Wnt3a-induced dedifferentiation of mature adipocytes produced reciprocal changes in P1 and P2 transcript levels. P2-derived gene-products containing frizzled-like sequences bound the potent adipogenic inhibitor, Wnt10b, in vitro. Previously, we have shown that these same sequences bind Wnt3a, inhibiting Wnt3a-mediated signaling. P2-transcript levels in visceral fat were positively correlated with serum free fatty acid levels, suggesting that collagen α1 (XVIII) expression contributes to regulation of adipose tissue metabolism in visceral obesity. Medium/long collagen XVIII is deposited in the Space of Disse, and interaction between hepatic apolipoprotein E and this proteoglycan is lost in P2-null mice. These results describe a previously unidentified extracellular matrix-directed mechanism contributing to the control of the multistep adipogenic program that determines the number of precursors committing to adipocyte differentiation, the maintenance of the differentiated state, and the physiological consequences of its impairment on ectopic fat deposition.

Lack of collagen XVIII long isoforms affects kidney podocytes, whereas the short form is needed in the proximal tubular basement membrane.

Collagen XVIII is characterized by three variant N termini, an interrupted collagenous domain, and a C-terminal antiangiogenic domain known as endostatin. We studied here the roles of this collagen type and its variant isoforms in the mouse kidney. Collagen XVIII appeared to be in a polarized orientation in the tubular basement membranes (BMs), the endostatin domain embedded in the BM, and the N terminus residing at the BM-fibrillar matrix interface. In the case of the glomerular BM (GBM), collagen XVIII was expressed in different isoforms depending on the side of the GBM. The orientation appeared polarized here, too, both the endothelial promoter 1-derived short variant of collagen XVIII and the epithelial promoter 2-derived longer variants having their C-terminal endostatin domains embedded in the BM and the N termini at the respective BM-cell interfaces. In addition to loosening of the proximal tubular BM structure, the Col18a1(-/-) mice showed effacement of the glomerular podocyte foot processes, and microindentation studies showed changes in the mechanical properties of the glomeruli, the Col18a1(-/-) glomeruli being ∼30% softer than the wild-type. Analysis of promoter-specific knockouts (Col18a1(P1/P1) and Col18a1(P2/P2)) indicated that tubular BM loosening is due to a lack of the shortest isoform, whereas the glomerular podocyte effacement was due to a lack of the longer isoforms. We suggest that lack of collagen XVIII may also have disparate effects on kidney function in man, but considering the mild physiological findings in the mutant mice, such effects may manifest themselves only late in life or require other compounding molecular changes.

Endostatin affects osteoblast behavior in vitro, but collagen XVIII/endostatin is not essential for skeletal development in vivo.

Endostatin, a fragment of collagen XVIII, can inhibit vascular endothelial growth factor (VEGF) signaling. VEGF is known to be crucial for bone development. The aims of this study were to investigate the influences of endostatin on osteoblast behavior in vitro and the roles of collagen XVIII/endostatin on bone development in vivo. For the in vitro experiments, MC3T3-E1 osteoblasts were treated with VEGF-A, 2 microg/ml endostatin, 20 microg/ml endostatin, VEGF-A + 2 microg/ml endostatin, or VEGF-A + 20 microg/ml endostatin. Osteoblast proliferation and matrix mineralization were analyzed. Faxitron, pQCT, and histological analyses were performed on hindleg bones of transgenic mice overexpressing endostatin (ES-tg) and mice lacking collagen XVIII (Col18a1 (-/-)) to study bone development in vivo. Treatment of cells with endostatin decreased osteoblast proliferation. Moreover, VEGF-A together with endostatin (2 microg/ml) decreased osteoblast proliferation and matrix mineralization. In vivo, Col18a1 (-/-) and ES-tg mice displayed no differences in bone density or mineral content during bone development, but ES-tg bones grew in length more slowly compared to the controls. The formation of secondary ossification centers was delayed in Col18a1 (-/-) mice. Immunohistochemistry revealed collagen XVIII in basement membranes of periosteal and bone marrow vessels and at muscle attachment sites. In conclusion, endostatin affects osteoblast behavior in vitro, the effects being boosted by simultaneous treatment with VEGF. In vivo, Col18a1 (-/-) and ES-tg mice show mild delays in bone development. These changes are transitory and suggest that collagen XVIII/endostatin does not play an indispensable role in skeletal development.

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing.

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.

The multiple functions of collagen XVIII in development and disease.

Collagen XVIII is a heparan sulphate proteoglycan which is expressed ubiquitously in different basement membranes throughout the body. Its C-terminal fragment, endostatin, has been found to inhibit angiogenesis and tumor growth by restricting endothelial proliferation and migration and inducing apoptosis of endothelial cells. Collagen XVIII has three variants, of which the shortest one is found in most vascular and epithelial BM structures, whereas the longer variants are found especially in the liver. The longest or frizzled variant has a cysteine-rich domain in its N-terminus that has been shown to inhibit Wnt signaling in vitro. The presence of collagen XVIII homologues in organisms such as C. elegans, Xenopus laevis, zebrafish and chick suggests a fundamental role for this BM collagen. Mutations in the collagen XVIII gene lead to the Knobloch syndrome, which is characterized by high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. Mice lacking collagen XVIII also show several ocular abnormalities. This suggests that in physiological conditions collagen XVIII is mostly needed for the proper development of the eye. Moreover, it appears to be needed for the structural stability of basement membranes in several other organs, and increasing evidence shows its importance for other organs in non-physiological situations such as atherosclerosis, glomerulonephritis or other type of tissue damage. This review focuses on clarifying the roles of collagen XVIII and its variants and domains in various physiological and pathological conditions.