Dries Bauters

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death

Our study underscores the importance of elastin fragmentation in the vessel wall as an accelerator of atherosclerosis with enhanced inflammation and increased neovascularization, thereby promoting the development of unstable plaques that eventually may rupture. The present mouse model offers the opportunity to further investigate the role of key factors involved in plaque destabilization and potential targets for therapeutic interventions.

Gelatinase A (MMP-2) promotes murine adipogenesis.

BACKGROUND Expansion of adipose tissue is dependent on adipogenesis, angiogenesis and extracellular matrix remodeling. A functional role in these processes was suggested for the gelatinase subfamily of the matrix metalloproteinases. Here, we have evaluated a potential role of gelatinase A (MMP-2) in adipogenesis. METHODS Murine embryonic fibroblasts (MEF) were derived from wild-type or MMP-2 deficient mice. Genetic manipulation of Mmp2 (shRNA-knockdown or overexpression) was performed in 3T3-F442A preadipocytes. Cell cultures were subjected to an adipogenic medium. As an in vivo model for de novo adipogenesis, 3T3-F442A preadipocytes with or without knockdown were injected subcutaneously in Nude BALB/c mice kept on high fat diet. RESULTS Mmp2 deficient MEF, as compared to controls, showed significantly impaired differentiation into mature adipocytes, as demonstrated by 90% reduced intracellular lipid content and reduced expression of pro-adipogenic markers. Moreover, selective Mmp2 knockdown in 3T3-F442A preadipocytes resulted in significantly reduced differentiation. In contrast, overexpression of Mmp2 resulted in markedly enhanced differentiation. In de novo formed fat pads resulting from preadipocytes with Mmp2 knockdown expression of aP2, Ppar-γ and adiponectin was significantly lower, and collagen was more preserved. The fat pad weights as well as size and density of adipocytes or blood vessels were, however, not significantly different from controls. CONCLUSION Our data directly support a functional role of MMP-2 in adipogenesis in vitro, and suggest a potential role in in vivo adipogenesis. GENERAL SIGNIFICANCE Selective modulation of MMP-2 levels affects adipogenesis.

Effect of gelatinase inhibition on adipogenesis and adipose tissue development

1. The potential of the matrix metalloproteinase (MMP) inhibitor ABT‐518 to affect pre‐adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using mouse models of adipogenesis and obesity.

The Gustatory Signaling Pathway and Bitter Taste Receptors Affect the Development of Obesity and Adipocyte Metabolism in Mice

Intestinal chemosensory signaling pathways involving the gustatory G-protein, gustducin, and bitter taste receptors (TAS2R) have been implicated in gut hormone release. Alterations in gut hormone profiles may contribute to the success of bariatric surgery. This study investigated the involvement of the gustatory signaling pathway in the development of diet-induced obesity and the therapeutic potential of targeting TAS2Rs to induce body weight loss. α-gustducin-deficient (α-gust-/-) mice became less obese than wild type (WT) mice when fed a high-fat diet (HFD). White adipose tissue (WAT) mass was lower in α-gust-/- mice due to increased heat production as a result of increases in brown adipose tissue (BAT) thermogenic activity, involving increased protein expression of uncoupling protein 1. Intra-gastric treatment of obese WT and α-gust-/- mice with the bitter agonists denatonium benzoate (DB) or quinine (Q) during 4 weeks resulted in an α-gustducin-dependent decrease in body weight gain associated with a decrease in food intake (DB), but not involving major changes in gut peptide release. Both WAT and 3T3-F442A pre-adipocytes express TAS2Rs. Treatment of pre-adipocytes with DB or Q decreased differentiation into mature adipocytes. In conclusion, interfering with the gustatory signaling pathway protects against the development of HFD-induced obesity presumably through promoting BAT activity. Intra-gastric bitter treatment inhibits weight gain, possibly by directly affecting adipocyte metabolism.

Gelatinase B (MMP-9) gene silencing does not affect murine preadipocyte differentiation

Growth of adipose tissue involves differentiation of preadipocytes into mature lipid-containing adipocytes. The matrix metalloproteinases (MMPs) are known regulators of adipose tissue biology, and previous studies suggested a key role for gelatinase B (MMP-9) in adipogenesis. In the present study we have evaluated a potential functional role of MMP-9 by performing gene silencing in 3T3-F442A preadipocytes. At the end of a 12-day differentiation period, no significant differences were observed between MMP-9 knockdown cells and the control cells with respect to intracytoplasmatic lipid content, or expression of the adipogenic markers aP2, PPARγ, Lpl, and adiponectin, or of the preadipocyte marker Pref-1. Thus, in vitro differentiation of 3T3-F442A preadipocytes into mature adipocytes is not significantly affected by the loss of MMP-9.

Macrophage elastase (MMP-12) in expanding murine adipose tissue.

BACKGROUND Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue. METHODS The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks. RESULTS MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue. CONCLUSIONS Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS. GENERAL SIGNIFICANCE MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.

Differential effects of a gelatinase inhibitor on adipocyte differentiation and adipose tissue development

A potential role for the gelatinases in adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using the gelatinase inhibitor tolylsam ((R)‐3‐methyl‐2‐[4‐(3‐p‐tolyl‐[1,2,4]oxadiazol‐5‐yl)‐benzenesulphonylamino]‐butyric acid). Differentiation of murine 3T3‐F442A preadipocytes (12 days after reaching confluence) into mature adipocytes in vitro was promoted in the presence of tolylsam (10–100 μmol/L). De novo development of fat tissue in nude mice injected with preadipocytes and kept on a high‐fat diet was significantly impaired following treatment with tolylsam (100 mg/kg per day for 4 weeks). Adipose tissue development in matrix metalloproteinase (MMP)‐2 deficient mice, kept on a high‐fat diet, was significantly impaired following administration of tolylsam (100 mg/kg per day for 15 weeks). This was associated with markedly enhanced metabolic rate. Treatment of MMP‐2‐deficient mice with tolylsam (100 mg/kg per day, 15 weeks) was associated with the preservation of collagen and a reduction in blood vessel size in adipose tissues in vivo. Furthermore, plasma levels of triglycerides and free fatty acids were reduced by tolylsam treatment of MMP‐2‐deficient mice (100 mg/kg per day, 15 weeks), whereas nutrient adsorption in the intestine was not affected. The results of the present study indicate that tolylsam promotes preadipocyte differentiation in vitro, but impairs adipose tissue development in vivo.

ADAMTS13 deficiency promotes microthrombosis in a murine model of diet-induced liver steatosis.

ADAMTS13 cleaves ultralarge multimeric von Willebrand Factor (VWF), thereby preventing formation of platelet-rich microthrombi. ADAMTS13 is mainly produced by hepatic stellate cells, and numerous studies have suggested a functional role of ADAMTS13 in the pathogenesis of liver diseases. The aim of our study was to investigate a potential role of ADAMTS13 in formation of hepatic microthrombi and development of non-alcoholic steatohepatitis (NASH), and furthermore to evaluate whether plasmin can compensate for the absence of ADAMTS13 in removal of thrombi. Therefore, we used a model of high-fat diet-induced steatosis in Adamts13 deficient (Adamts13-/-) and wild-type (WT) control mice. Microthrombi were more abundant in the liver of obese Adamts13-/- as compared to obese WT or to lean Adamts13-/- mice. Obese Adamts13-/- mice displayed lower platelet counts and higher prevalence of ultra-large VWF multimers. Hepatic plasmin-α2-antiplasmin complex levels were comparable for obese WT and Adamts13-/- mice and were lower for lean Adamts13-/- than WT mice, not supporting marked activation of the fibrinolytic system. High fat diet feeding, as compared to normal chow, resulted in enhanced liver triglyceride levels for both genotypes (p < 0.0001) and steatosis (p < 0.0001 for WT mice, p = 0.002 for Adamts13-/- mice) without differences between the genotypes. Expression of markers of inflammation, oxidative stress, steatosis and fibrosis was affected by diet, but not by genotype. Thus, our data confirm that obesity promotes NASH, but do not support a detrimental role of ADAMTS13 in its development. However, Adamts13 deficiency in obese mice promotes hepatic microthrombosis, whereas a compensatory role of plasmin in removal of microthrombi in the absence of ADAMTS13 could not be demonstrated.

ADAMTS5 deficiency protects against non-alcoholic steatohepatitis in obesity

Increased prevalence of obesity is paralleled by an increase in non‐alcoholic steatohepatitis (NASH). We previously found that the expression of ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) is enhanced in expanding adipose tissue. However, no information is available on a potential role in liver pathology. We studied the effect of ADAMTS5 deficiency on NASH in mice.

ADAMTS5 promotes murine adipogenesis and visceral adipose tissue expansion

Enhanced expression of the aggrecanase ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) has been observed in adipose tissue (AT) of obese rodents. Here, we have investigated the role of ADAMTS5 in adipogenesis, AT expansion and associated angiogenesis. In vitro differentiation of precursor cells into mature adipocytes was studied using murine embryonic fibroblasts (MEF) derived from wild-type (Adamts5(+/+)) and ADAMTS5 deficient (Adamts5(-/-)) mice, or 3T3-F442A preadipocytes with stable gene silencing of Adamts5. De novo adipogenesis was monitored by injection of 3T3-F442A cells with or without Adamts5 knockdown in Nude mice. Furthermore, Adamts5(+/+)and Adamts5(-/-) mice were kept on a high-fat diet (HFD) to monitor AT development. Adamts5(-/-) MEF, as well as 3T3-F442A preadipocytes with Adamts5 knockdown, showed significantly reduced differentiation as compared to control cells. In mice, de novo formed fat pads arising from 3T3-F442A cells with Adamts5 knockdown were significantly smaller as compared to controls. After 15 or 25 weeks on HFD, total body weight and subcutaneous AT weight were similar for Adamts5(+/+) and Adamts5(-/-) mice, but visceral/gonadal fat mass was significantly lower for Adamts5(-/-) mice. These data were confirmed by magnetic resonance imaging. In addition, the blood vessel density in adipose tissue was higher for Adamts5(-/-) mice kept on HFD. In conclusion, our data support the concept that ADAMTS5 promotes adipogenesis in vitro and in vivo, as well as development of visceral AT and associated angiogenesis in mice kept on HFD.