Ilse Scroyen

Impaired Adipose Tissue Development in Mice With Inactivation of Placental Growth Factor Function

Placental growth factor (PlGF)-deficient (PlGF−/−) and wild-type mice were kept on a standard-fat or high-fat diet for 15 weeks. With the standard-fat diet, the body weights of PlGF−/− and wild-type mice were comparable, whereas the combined weight of subcutaneous and gonadal adipose tissues was lower in PlGF−/− mice (P = 0.02). With the high-fat diet, PlGF−/− mice had a lower body weight (P < 0.05) and less total subcutaneous plus gonadal adipose tissue (P < 0.0001). Blood vessel size was lower in gonadal adipose tissue of PlGF−/− mice with both the standard-fat and high-fat diet (P < 0.05). Blood vessel density, normalized to adipocyte number, was significantly lower in subcutaneous adipose tissue of PlGF−/− mice fed the high-fat diet (P < 0.01). De novo adipose tissue development in nude mice injected with 3T3-F442A preadipocytes was reduced (P < 0.005) by administration of a PlGF-neutralizing antibody. Bone marrow transplantation from wild-type or PlGF−/− mice to wild-type or PlGF−/− recipient mice revealed significantly lower blood vessel density in PlGF−/− recipient mice without an effect on adipose tissue growth. Thus, in murine models of diet-induced obesity, inactivation of PlGF impairs adipose tissue development, at least in part as a result of reduced angiogenesis.

CD36 promotes adipocyte differentiation and adipogenesis.

BACKGROUND CD36 is a membrane glycoprotein, contributing to the pathogenesis of metabolic disorders, like obesity, which has become a major health concern worldwide. METHODS A potential functional role of the scavenger receptor CD36 was investigated in in vitro adipocyte differentiation and in vivo adipogenesis. RESULTS During differentiation of 3T3-F442A preadipocytes into mature adipocytes, expression of CD36 was upregulated and CD36 gene silencing resulted in impaired differentiation, as monitored by Oil Red O staining and expression of adipogenic markers. De novo fat pad formation in NUDE mice following injection of preadipocytes was significantly reduced upon CD36 gene silencing as compared to control. This was associated with marked adipocyte hypotrophy and reduced adipose tissue adipocyte content. Macrophage infiltration in de novo fat tissues derived from preadipocytes with CD36 gene silencing was not significantly different from controls. Collagen content was significantly higher in de novo fat with CD36 gene silencing. In a nutritionally induced obesity model, total body weight as well as subcutaneous and gonadal adipose tissue mass were significantly lower in CD36 deficient mice as compared to wild-type littermates. GENERAL SIGNIFICANCE Thus, our data support a functional role of CD36 in promoting adipogenesis in vitro as well as in vivo.

Role of proteolysis in development of murine adipose tissue

Obesity is a common disorder, and related diseases such as diabetes, atherosclerosis, hypertension, cardiovascular disease and cancer are a major cause of mortality and morbidity in Western-type societies. Development of obesity is associated with extensive modifications in adipose tissue involving adipogenesis, angiogenesis and extracellular matrix proteolysis. The fibrinolytic (plasminogen/plasmin) and matrix metalloproteinase (MMP) systems cooperate in these processes. A nutritionally induced obesity model in transgenic mice has been used extensively to study the role of the fibrinolytic and MMP systems in the development of obesity. These studies support a role of both systems in adipogenesis and obesity, and suggest that modulation of proteolytic activity may affect development of adipose tissue.

MicroRNAs regulated by adiponectin as novel targets for controlling adipose tissue inflammation.

A low-grade proinflammatory state contributes to the metabolic syndrome (MS). Adiponectin (ApN), which is reduced in the MS, has emerged as a master regulator of inflammation/immunity. We wanted to identify whether microRNAs (miRNAs) may mediate the antiinflammatory action of ApN on adipose tissue (AT). miRNA expression profiling was performed in mice overexpressing ApN specifically in AT and in wild-type controls. The role of specific miRNAs was analyzed by gain- or loss-of function approaches in 3T3-F442A (pre)-adipocytes and in de novo AT formed from engineered 3T3-F442A preadipocytes transplanted in nude mice. miRNA expression was compared in the omental AT of lean and obese subjects. The expression of miR532-5p and miR1983 was down-regulated, whereas that of miR883b-5p and miR1934 was up-regulated in AT of mice overexpressing ApN specifically in AT. We focused on miR883b-5p identified by computational analysis as being involved in inflammatory pathways. miR883b-5p overexpression down-regulated the lipopolysaccharide-binding protein (LBP) in 3T3-F442A cells, whereas miR883b-5p blockade had reverse effects. LBP aids in lipopolysaccharide binding to Toll-like receptor-4. miR883b-5p blockade also abolished the protective effects of ApN on proinflammatory adipokine induction. These data were recapitulated in the de novo AT in which miR883b-5p silencing induced LBP production and tissue inflammation. Eventually miR883b-5p expression was down-regulated in AT of obese subjects. We identified several novel miRNAs that are regulated by ApN in AT in vivo. miR883b-5p, which is up-regulated by ApN represses LBP and Toll-like receptor-4 signaling, acting therefore as a major mediator of the antiinflammatory action of ApN. These novel miRNAs may open new therapeutic perspectives for the MS.

Deficiency of plasminogen activator inhibitor‐2 impairs nutritionally induced murine adipose tissue development

Background: A functional role for several components of the fibrinolytic (plasminogen/plasmin) system in development of adipose tissue has been demonstrated. No information is available, however, on a potential role of plasminogen activator inhibitor‐2 (PAI‐2) in obesity. Methods: In vitro, 3T3‐F442A murine pre‐adipocytes were differentiated into mature adipocytes. In vivo, 5‐week‐old male PAI‐2‐deficient (PAI‐2−/−) mice and wild‐type (WT) controls of the same genetic background (C57Bl/6) were kept on a high fat diet (HFD, caloric value of 20.1 kJ g−1) for 15 weeks. Results: Semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) revealed expression of PAI‐2 mRNA during in vitro differentiation of pre‐adipocytes and in vivo in s.c. and gonadal (GON) adipose tissues of WT mice, where it was localized both in the stromal/vascular cell fraction and in adipocytes. During HFD feeding, food intake and body weight gain were comparable for WT and PAI‐2−/− mice. Subcutaneous plus GON fat mass was, however, significantly lower in PAI‐2−/− mice (3.15 ± 0.21 vs. 3.91 ± 0.18 g; P < 0.05). Immunohistochemical analysis of adipose tissues revealed significant adipocyte hypotrophy in s.c. fat of PAI‐2−/− mice (about 25% reduction in size; P < 0.01). Blood vessel density, normalized to adipocyte number, was comparable in s.c. fat, but was lower (P < 0.05) in GON fat of PAI‐2−/− mice. Adipose tissue‐associated fibrinolytic activity was not affected by PAI‐2 deficiency. Conclusion: PAI‐2 promotes adipose tissue development in mice via a mechanism independent of its antifibrinolytic effect.

Effect of tissue inhibitor of matrix metalloproteinases-1 on in vitro and in vivo adipocyte differentiation

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a role in development of obesity by contributing to adipogenesis, angiogenesis and extracellular matrix degradation. We have evaluated a potential functional role of TIMP-1, which inhibits most MMPs, in early stages of in vitro and in vivo adipogenesis. Overexpression of human TIMP-1 (hTIMP-1) in 3T3-F442A preadipocytes resulted in a somewhat slower differentiation into mature adipocytes, without affecting the total extent of differentiation. Local overexpression by injection of 3T3-F442A preadipocytes expressing hTIMP-1 in the back of NUDE mice kept on high fat diet (HFD) for 4 weeks, had no effect on de novo formed fat pad mass. The fat pads formed from the hTIMP-1 expressing cells did show a significantly larger blood vessel size as compared to control cells (57 + or - 4.8 microm(2) vs 38 + or - 1.4 microm(2), p=0.0017). No effect was observed on blood vessel density or on adipocyte size or density. Thus, local hTIMP-1 overexpression did not significantly affect early stages of adipogenesis as evaluated from the extent of in vitro adipocyte differentiation or of in vivo de novo adipose tissue formation.

Overexpression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in mice does not affect adipogenesis or adipose tissue development.

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.

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.

Maximal PAI-1 inhibition in vivo requires neutralizing antibodies that recognize and inhibit glycosylated PAI-1

Plasminogen activator inhibitor-1 (PAI-1) regulates the activity of t-PA and u-PA and is an important inhibitor of the plasminogen activator system. Elevated PAI-1 levels have been implicated in the pathogenesis of several diseases. Prior to the evaluation of PAI-1 inhibitors in humans, there is a strong need to study the effect of PAI-1 inhibition in mouse models. In the current study, four monoclonal antibodies previously reported to inhibit recombinant PAI-1 in vitro, were evaluated in an LPS-induced endotoxemia model in mice. Both MA-33H1F7 and MA-MP2D2 exerted a strong PAI-1 inhibitory effect, whereas for MA-H4B3 and MA-124K1 no reduced PAI-1 activity was observed in vivo. Importantly, the lack of PAI-1 inhibition observed for MA-124K1 and MA-H4B3 in vivo corresponded with the absence of inhibition toward glycosylated mouse PAI-1 in vitro. Three potential N-glycosylation sites were predicted for mouse PAI-1 (i.e. N209, N265 and N329). Electrophoretic mobility analysis of glycosylation knock-out mutants before and after deglycosylation indicates the presence of glycan chains at position N265. These data demonstrate that an inhibitory effect toward glycosylated PAI-1 is a prerequisite for efficient PAI-1 inhibition in mice. Our data also suggest that PAI-1 inhibitors for use in humans must preferably be screened on glycosylated PAI-1 and not on recombinant non-glycosylated PAI-1.

No functional role of plasminogen activator inhibitor-1 in murine adipogenesis or adipocyte differentiation.

Summary.  Objective: To substantiate a potential role of plasminogen activator inhibitor‐1 (PAI‐1) in adipogenesis, we have studied its effects on in vitro adipocyte differentiation and on in vivo adipose tissue formation. Results: Our in vitro data do not support a functional role of PAI‐1, as substantiated by our findings that: (i) inhibition of PAI‐1 with a neutralizing antibody did not affect differentiation of 3T3‐F442A preadipocytes; (ii) overexpression of murine PAI‐1 in 3T3‐F442A cells had no effect on differentiation; and (iii) differentiation of PAI‐1‐deficient murine embryonic fibroblasts into mature adipocytes was comparable to wild‐type (WT) cells. Furthermore, our in vivo studies did not reveal an important role for PAI‐1, as suggested by our findings that: (i) de novo fat pad formation in NUDE mice following injection of 3T3‐F442A cells was not affected by a PAI‐1 neutralizing antibody; and (ii) adipose tissue formation following combined injection of Matrigel and basic fibroblast growth factor was comparable in WT and in PAI‐1 deficient mice. Conclusion: Taken together, these in vitro and in vivo studies in murine model systems do not support an important functional role of PAI‐1 in adipogenesis.