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The potential role of the matrix metalloproteinase (MMP) system in the pathophysiology of the adipose tissue was investigated in a mouse model of nutritionally induced obesity. mRNA levels of 16 MMPs and 4 tissue inhibitors of MMPs (TIMPs) were measured by semiquantitative RT-PCR in adipose tissue isolated from mice maintained for 15 weeks on a standard or high-fat diet. In mice on standard diet, with the exception of MMP-8, all MMP and TIMP transcripts were detected in both gonadal and subcutaneous depots. In obese mice, the expression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was upregulated, whereas that of MMP-7, -9, -16, and -24 and TIMP-4 was downregulated. Most MMP and TIMP mRNAs were expressed at higher levels in stromal-vascular cells than in mature adipocytes. Analysis of adipose tissue by in situ fluorescent zymography revealed MMP-dependent proteolytic activities, demonstrating the presence of active MMPs in the intact tissue. In vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes was associated with substantial modulations of MMP and TIMP expression. Moreover, this in vitro adipogenesis was reduced in the presence of a synthetic MMP inhibitor. Thus, the adipose tissue expresses a large array of MMPs and TIMPs, which modulate adipocyte differentiation.

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.

BackgroundMicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated.Methodology/Principal FindingsWe first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization.Conclusions/SignificanceOur results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription-polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post-translational enzymes, the carboxy- and amino-procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased.