Development of vasculature and mRNA expression of 17 pro- or antiangiogenic factors were studied during adipose tissue development in nutritionally induced or genetically determined murine obesity models. Subcutaneous (SC) and gonadal (GON) fat pads were harvested from male C57Bl/6 mice kept on standard chow [standard fat diet (SFD)] or on high-fat diet for 0-15 wk and from male ob/ob mice kept on SFD. Ob/ob mice and C57Bl/6 mice on high-fat diet had significantly larger SC and GON fat pads, accompanied by significantly higher blood content, increased total blood vessel volume, and higher number of proliferating cells. mRNA and protein levels of angiopoietin (Ang)-1 were down-regulated, whereas those of thrombospondin-1 were up-regulated in developing adipose tissue in both obesity models. Ang-1 mRNA levels correlated negatively with adipose tissue weight in the early phase of nutritionally induced obesity as well as in genetically determined obesity. Placental growth factor and Ang-2 expression were increased in SC adipose tissue of ob/ob mice, and thrombospondin-2 was increased in both their SC and GON fat pads. mRNA levels of vascular endothelial growth factor (VEGF)-A isoforms VEGF-B, VEGF-C, VEGF receptor-1, -2, and -3, and neuropilin-1 were not markedly modulated by obesity. This modulation of angiogenic factors during development of adipose tissue supports their important functional role in obesity.

SummaryTo investigate a potential role of stromelysin-1 (MMP-3) in development of adipose tissue, 5 week old male MMP-3 deficient mice (MMP-3-/-) and wild-type (MMP-3+/+) controls were kept on a high fat diet (HFD) for 15 weeks. MMP-3-/- mice were hyperphagic and gained more weight than the MMP-3+/+ mice. At the time of sacrifice, the body weight of the MMP-3-/- mice was significantly higher than that of the MMP-3+/+ mice, as was the weight of the isolated subcutaneous (SC) and gonadal (GON) fat deposits. Significant adipocyte hypertrophy was observed in the GON but not in the SC adipose tissue of MMP-3-/- mice. Fasting plasma glucose and cholesterol levels were comparable in both genotypes, whereas triglyceride levels were significantly lower in MMP-3-/- mice. Staining with an endothelial cell specific lectin revealed a significantly higher blood vessel density and larger total stained area in the GON adipose tissues of MMP-3-/- mice. Thus, in a murine model of nutritionally induced obesity, MMP-3 impairs adipose tissue development, possibly by affecting food intake and/or adipose tissue-related angiogenesis.Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.

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In order to evaluate a potential functional role of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in development of obesity, we studied the effect of overexpression of human TIMP-1 (hTIMP-1) in C57Bl/6J mice in vivo and in 3T3-F442A preadipocytes in vitro. Stable long-term overexpression of hTIMP-1 in mice was achieved by adenoviral gene transfer, yielding plasma levels exceeding 250 ng/ml at eight weeks after injection. Mice overexpressing hTIMP-1 and kept on a high fat diet for 14 weeks had body weights, adipose tissue weights, and adipocyte diameters that were somewhat, but not significantly, lower than those of control mice. Similar observations were made after overexpression of hTIMP-1 in mice with lipectomy of the subcutaneous adipose tissue, kept on a high fat diet for 20 weeks. In both in vivo models, blood vessels in the adipose tissues were significantly smaller after hTIMP-1 gene transfer than in control mice. Overexpression of hTIMP-1 in 3T3-F442A preadipocytes had no effect on their subsequent differentiation into mature adipocytes. Thus, overexpression of hTIMP-1 in mice had no significant effect on ongoing adipogenesis or adipose tissue development, although the blood vessel size in adipose tissues was reduced.

Thromb Haemost 2003; 89: 249 -55TIMP-1 -/-mice. Plasma leptin levels in TIMP-1 -/-mice on HFD were significantly lower as compared to TIMP-1 +/-mice, and strongly correlated with adipose tissue mass for both genotypes. Staining with an endothelial cell specific lectin revealed a significantly higher blood vessel density, larger stained area and vessel size in adipose tissue of TIMP-1 -/-mice on HFD. This difference disappeared after normalization to the adipocyte number, suggesting that it does not represent a true enhancement of angiogenesis. Thus, in a murine model of nutritionally induced obesity,TIMP-1 promotes adipose tissue development. KeywordsTissue inhibitor of metalloproteinase-1,TIMP-1, adipose tissue, obesity, angiogenesis SummaryTissue inhibitor of matrix metalloproteinase-1 deficient (TIMP-1 -/-) mice and wild-type (TIMP-1 +/+ ) controls were kept on a standard (SFD) or a high fat diet (HFD) for 15 weeks. At the time of sacrifice,TIMP-1 -/-mice on HFD had a significantly lower body weight (29 ± 1.5 versus 41 ± 1.8 g, p <0.005), and significantly less subcutaneous (0.81 ± 0.19 versus 1.78 ± 0.21 g, p <0.05) and gonadal (0.87 ± 0.17 versus 1.85 ± 0.18 g, p <0.005) fat mass. These differences were much less pronounced for mice on SFD. On HFD but not on SFD, adipocyte diameters were significantly lower in the adipose tissue of

SummaryA nutritionally induced obesity model was used to investigate the modulation of fibrinolytic and gelatinolytic activity during the development of adipose tissue.Five week old male mice were fed a standard fat diet (SFD, 13% kcal as fat) or a high fat diet (HFD, 42% kcal as fat) for up to 15 weeks. The HFD resulted in body weights of 31 ± 0.9 g, 38 ± 2.0 g and 47 ± 1.9 g at 5, 10 and 15 weeks, respectively; corresponding values for mice on the SFD were 26 ± 0.6 g, 31 ± 0.9 g and 31 ± 1.2 g (all p < 0.001). The weight of the isolated subcutaneous (SC) or gonadal (GON) fat after 15 weeks of HFD was 1,870 ± 180 mg or 1,470 ± 160 mg, as compared to 250 ± 58 mg or 350 ± 71 mg for the SFD (p < 0.001). The HFD induced marked time-dependent hyperglycemia and elevated levels of triglycerides and total cholesterol. The HFD diet also induced a marked hypertrophy of the adipocytes as compared to the SFD, e.g. diameter of 83 ± 3.0 µm versus 52 ± 4.2 µm for GON adipocytes at 15 weeks (p < 0.005). Plasma plasminogen activator inhibitor-1 (PAI-1) levels were higher in mice on the HFD as compared to the SFD; they were comparable in extracts of SC or GON adipose tissue, whereas at different time points tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator activity was somewhat lower in the adipose tissues of mice on HFD. Gelatinolytic activity (mainly MMP-2) was detected in SC but not in GON adipose tissue of mice on SFD, and decreased on the HFD. In situ zymography on cryosections did not reveal different fibrinolytic activities in SC or GON adipose tissues of the HFD as compared to the SFD groups, whereas significantly lower gelatinolytic and higher caseinolytic activities were detected in SC and GON tissues of mice on the HFD (p ≤ 0.05). The fibrillar collagen content was lower in adipose tissue of mice on HFD. Thus, in this model time-dependent development of adipose tissue appears to be associated with modulation of proteolytic activity.

Matrix metalloproteinases (MMPs) may play a role in development of obesity by contributing to adipogenesis, angiogenesis and extracellular matrix degradation. To evaluate a potential functional role of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1, which inhibits most MMPs), we have overexpressed human TIMP-1 (hTIMP-1) in C57Bl/6J mice in vivo and in 3T3-F442A preadipocytes in vitro. Stable long-term overexpression of hTIMP-1 in mice was achieved by adenoviral gene transfer, yielding plasma levels exceeding 250 ng/ml at 8 weeks after injection. Mice overexpressing hTIMP-1 (n= 15) and kept on high fat diet for 14 weeks had body weight (31 ± 0.8 versus 33 ± 0.5 g), subcutaneous (SC) (0.61 ± 0.08 versus 0.73 ± 0.08 g) and gonadal (GON) (1.01 ± 0.11 versus 1.24 ± 0.08 g) adipose tissue weights, and adipocyte diameters (26 ± 3 versus 28 ± 3 µm for SC, and 35 ± 4 versus 37 ± 4 µm for GON) that were somewhat, but not significantly, lower than control mice (n= 16). Reduced MMP activity following hTIMP-1 gene transfer was confirmed by gelatin zymography using adipose tissue extracts and by in situ zymography using gelatin overlays on cryosections. Expression of murine TIMP-1, MMP-2 and MMP-9 mRNA in adipose tissues was not altered after hTIMP-1 gene transfer. Physical activity and food intake were comparable in both groups. Metabolic parameters, including glucose, cholesterol and triglycerides were not affected by overexpression of hTIMP-1. Histological analysis revealed that the blood vessels in adipose tissues of mice overexpressing hTIMP-1 were significantly smaller than in controls (39 ± 4.0 versus 49 ± 3.3 µm2 for SC, p < 0.001; 37 ± 3.3 versus 61 ± 3.8 µm2 for GON, p < 0.01). Similar observations were made after overexpression of hTIMP-1 in mice with lipectomy of the SC adipose tissue, kept on high fat diet for 20 weeks. No significant effects of hTIMP-1 gene transfer were observed on total body weights (41 ± 1.0 versus 41 ± 0.3 g in control mice (n= 5 or 6)), or on weights of the isolated SC (0.84 ± 0.08 versus 0.89 ± 0.05 g) or GON (2.8 ± 0.08 versus 2.8 ± 0.05 g) adipose tissues. Blood vessels were significantly smaller in hTIMP-1 overexpressing adipose tissues (14 ± 1 versus 19 ± 1 µm2 for SC, p < 0.01; 18 ± 1 versus 23 ± 2 µm2 for GON, p < 0.001). Overexpression of hTIMP-1 in 3T3-F442A preadipocytes in vitro (pIRES2 EGFP) had no effect on their subsequent differentiation into mature adipocytes, as judged from Oil Red 0 staining. Taken together, these studies reveal that overexpression of hTIMP-1 in mice reduces blood vessel size in adipose tissues without, however, a significant effect on adipogenesis or adipose tissue development. These findings thus seem to challenge the relative importance of angiogenesis in ongoing adipose tissue development.