LPS Counter Regulates RNA Expression of Extracellular Proteases and Their Inhibitors in Murine Macrophages

Besides their evident importance in host defense, macrophages have been shown to play a detrimental role in different pathological conditions, including chronic inflammation, atherosclerosis, and cancer. Regardless of the exact situation, macrophage activation and migration are intimately connected to extracellular matrix degradation. This process is accomplished by multiple proteolytic enzymes, including serine proteases and members of the matrix metalloproteinase family. In this study, we have utilized qPCR arrays to simultaneously analyze the temporal expression pattern of a range of genes involved in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario was found for the genes encoding serine proteases, which were downregulated while their inhibitors were upregulated. In addition, intergenic comparison of the expression levels of related proteases revealed large differences in their basal expression level. These data highlight the complexity of the gene expression regulation implicated in macrophage-dependent matrix degradation and furthermore emphasize the value of qPCR array techniques for the investigation of the complex regulation of the matrix degradome

Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

Background: Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds. Methodology/Principal Findings: To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure. Conclusions/Significance: Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murin

MMP9 is protective against lethal inflammatory mass lesions in the mouse colon

The family of matrix metalloproteinases (MMPs) is responsible for extracellular matrix degradation during physiological and pathophysiological tissue remodeling processes such as embryogenesis, tissue repair and cancer progression. Despite these important roles of MMPs, inhibition or ablation of individual members of the MMP family in animal models have been shown to have little effect. It has been speculated that this results from a functional overlap between individual MMPs and (as-yet-unclassified) functional overlaps between MMPs and other protease systems. We here present genetic data showing that concomitant ablation of MMP9 (gelatinase B) and the serine protease plasmin results in lethal inflammatory mass lesions in the colon. These lesions possessed several histological attributes that are characteristic of mucosal prolapse seen in humans, and they were found to be associated with splenomegaly, enlarged mesenteric lymph nodes, decreased thymus size and altered populations of circulating immune cells. A time-course study provided evidence that the massive lymphoid hyperplasia and reactive changes were secondary to discrete fibrinous lesions also observed in mice only deficient for plasminogen (Plg), the zymogen for plasmin. These data demonstrate a non-appreciated vital protective role for MMP9 in the absence of Plg

Decreased cell infiltration in fibrin rich areas of wound tissues.

<p>Fibrin(ogen) staining of skin wound sections. The epidermis is underlined by green, fibrin(ogen) is stained brown with NovaRED, and nuclei are stained blue with a hematoxylin counter stain. (A–D) representative stainings of wound tissues from Wt and Plg^−/− mice of both genders. (E & F) enlarged micrographs of areas boxed in B. (G & H) sections from 50% healed wounds in the back skin of male mice, which show the area between the advancing keratinocytes and the underlying muscle tissue Note the lack of cells in this region in the Plg^−/− mice. (I) quantifications of the total area of fibrin(ogen) positive lesions in the provisional matrix and quantifications of the number of cells within and outside this area. n = 9–11 for each group.</p

Ovariectomy does not induce a male like phenotype in plasminogen deficient female mice.

<p>A possible effect of ovarian derived hormones on skin wound healing in Plg^−/− mice was tested by excision of the ovaries before a 20 mm full thickness incisional skin wound was made. (A) Wound lengths were measured using digital calipers every other day until completely healed. No statistical significant difference was found between the different groups belonging to Wt mice. The difference between sham operated male and female Plg^−/− mice were statistically significant (p<0.05), while no significant difference was found between OVX operated Plg^−/− mice and the sham operated Plg^−/− mice. (B) An area under the curve analysis showed no significant effect of OVX in Plg^−/− mice, while the difference between genders was significant. (C) The percentage of mice with completely reepithelialized wounds presented as a function of time. The bar graph shows the average time to complete reepithelialization.</p

The length of the hyperplastic epidermis and dermal wound gap is affected by gender.

<p>Histological analysis of H&E stained sections from fully reepithelialized wounds. (A–D) representative sections of healed wounds from Wt and Plg^−/− mice of both genders. The hyperplastic epidermis is underlined by green. (E & F) The length and average thickness of the hyperplastic epidermis. n = 9–11 for each group.</p

Gender does not affect the vessel concentration in fully reepithelialized wounds.

<p>CD34 stainings of fully reepithelialized wounds were performed to identify new vessels. (A–D) CD34 positive vessels were detected in the dermis proximal to the hyperplastic epidermis both in Wt and Plg^−/− mice of both genders. (E) Computerized staining analysis on whole wound sections reveal that Plg^−/− mice had a tendency to express less CD34 (area wise) than Wt mice while gender did not affect the level of CD34 staining.</p

Plasminogen deficiency does not affect the structure of naïve skin.

<p>The effect of Plg deficiency on the known differences in skin histology between genders was investigated using back skin from naïve Plg^−/− mice and Wt controls (8–12 weeks of age). n = 8–12 for each group. (A) The thickness of the dermis and hypodermis was determined by microscopy. (B) Representative skin sections from eight week old male and female mice.</p