Publications by authors named "Roswitha Nischt"

35 Publications

Dual role of laminin‑511 in regulating melanocyte migration and differentiation.

Matrix Biol 2019 07 29;80:59-71. Epub 2018 Sep 29.

Translational Matrix Biology, Medical Faculty, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. Electronic address:

Laminins are the major basement membrane (BM) components and are heterotrimers composed of an α, a β and a γ chain. In skin, laminins are present in basement membranes surrounding vascular structures, nerves, adipose tissue and in the specialized junctional BM between the epidermis and dermis. The main laminin isoforms in the dermo-epidermal BM are laminin‑332, laminin‑511 and laminin‑211, the latter being restricted to hair follicles (HFs). The laminin γ1 chain is the most abundant γ chain; its global ablation in mice leads to early embryonic lethality at E5.5. To elucidate the cellular function of the γ1 chain in skin, we generated mice with keratinocyte-specific deletion of this chain (Lamc1) by using the keratin (K)14-Cre/loxP system. These mice showed delayed coat pigmentation despite normal melanocyte counts in the skin. However, levels of differentiation-specific melanocyte enzymes TRP‑1, TRP‑2 and tyrosinase were reduced in Lamc1 mice, and melanocytes failed to migrate to their differentiation niche in HFs and accumulated in the IFE. These results suggested that the pigmentation defect results from impaired melanocyte migration. The impaired migratory capacity of melanocytes is due to the altered composition of laminins in the BM of Lamc1 mice: Loss of keratinocyte-derived pro-migratory laminin‑511 is not compensated by ectopically deposited fibroblast-derived laminin‑211. Furthermore, contact of melanocytes with recombinant laminin‑511, but not with laminin‑211, induces the expression of the chemokine receptor CXCR4 on melanocytes, needed for SDF‑1 (stromal cell‑derived factor‑1)-mediated migration into HFs. We here demonstrate that laminin‑511 controls the differentiation of melanocytes by regulating their migration from the epidermis into HFs and by activating CXCR4 expression on melanocytes required for their recruitment into HFs in an SDF‑1-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2018.09.006DOI Listing
July 2019

Deletion of the epidermis derived laminin γ1 chain leads to defects in the regulation of late hair morphogenesis.

Matrix Biol 2016 12 25;56:42-56. Epub 2016 May 25.

Dermatology, University of Cologne, Germany.

Laminins are the most abundant non-collagenous basement membrane (BM) components, composed of an α, β and γ chain. The laminin γ1 chain, encoded by LAMC1, is the most abundant γ chain. The main laminin isoforms in the dermo-epidermal junction (DEJ) are laminin-332, laminin-511 and laminin-211, the latter being restricted to the lower part of hair follicles (HFs). Complete deletion of LAMC1 results in lethality around embryonic day 5.5. To study the function of laminin γ1 containing isoforms in skin development and maturation after birth, we generated mice lacking LAMC1 expression in basal keratinocytes (LAMC1) using the keratin 14 (K14) Cre/loxP system. This deletion resulted in loss of keratinocyte derived laminin-511 and in deposition of fibroblast derived laminin-211 throughout the whole DEJ. The DEJ in areas between hemidesmosomes was thickened, whereas hemidesmosome morphology was normal. Most strikingly, LAMC1 mice showed delayed HF morphogenesis accompanied by reduced proliferation of hair matrix cells and impaired differentiation of hair shafts (HS). However, this deletion did not interfere with early HF development, since placode numbers and embryonic hair germ formation were not affected. Microarray analysis of skin revealed down regulation of mainly different hair keratins. This is due to reduced expression of transcription factors such as HoxC13, FoxN1, FoxQ1 and Msx2, known to regulate expression of hair keratins. While the role of laminin-511 in signaling during early hair germ formation and elongation phase has been described, we here demonstrate that epidermal laminin-511 is also a key regulator for later hair development and HS differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2016.05.002DOI Listing
December 2016

Laminin α5 in the keratinocyte basement membrane is required for epidermal-dermal intercommunication.

Matrix Biol 2016 12 24;56:24-41. Epub 2016 May 24.

Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, Germany. Electronic address:

Laminin α5 is broadly expressed in the epidermal basement membrane (BM) of mature mice and its elimination at this site (Lama5 mouse) results in hyperproliferation of basal keratinocytes and a delay in hair follicle development, which correlated with upregulation of the dermally-derived laminin α2 and laminin α4 chains in the epidermal BM and of tenascin-C subjacent to the BM. In vitro studies revealed laminin 511 to be strongly adhesive for primary keratinocytes and that loss of laminin α5 does not result in cell autonomous defects in proliferation. Flow cytometry reveals that the loss of laminin α5 resulted in increased numbers of CD45, CD4 and CD11b immune cells in the skin, which temporo-spatial analyses revealed were detectable only subsequent to the loss of laminin α5 and the appearance of the hyperproliferative keratinocyte phenotype. These findings indicate that immune cell changes are the consequence and not the cause of keratinocyte hyperproliferation. Loss of laminin α5 in the epidermal BM was also associated with changes in the expression of several dermally-derived growth factors involved in keratinocyte proliferation and hair follicle development in adult but not new born Lama5 skin, including KGF, EGF and KGF-2. In situ binding of FGF-receptor-2α (IIIb)-Fc chimera (FGFR2IIIb) to mouse skin sections revealed decoration of several BMs, including the epidermal BM, which was absent in Lama5 skin. This indicates reduced levels of FGFR2IIIb ligands, which include KGF and KGF-2, in the epidermal BM of adult Lama5 skin. Our data suggest an initial inhibitory effect of laminin α5 on basal keratinocyte proliferation and migration, which is exacerbated by subsequent changes in growth factor expression by epidermal and dermal cells, implicating laminin α5 in epidermal-dermal intercommunication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2016.05.001DOI Listing
December 2016

Tetanus toxin entry. Nidogens are therapeutic targets for the prevention of tetanus.

Science 2014 Nov;346(6213):1118-23

Sobell Department of Motor Neuroscience and Movement Disorders, University College London Institute of Neurology, University College London, London WC1N 3BG, UK.

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in nonvaccinated areas. TeNT targets the neuromuscular junction (NMJ) with high affinity, yet the nature of the TeNT receptor complex remains unknown. Here, we show that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Inhibition of the TeNT-nidogen interaction by using small nidogen-derived peptides or genetic ablation of nidogens prevented the binding of TeNT to neurons and protected mice from TeNT-induced spastic paralysis. Our findings demonstrate the direct involvement of an extracellular matrix protein as a receptor for TeNT at the NMJ, paving the way for the development of therapeutics for the prevention of tetanus by targeting this protein-protein interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1258138DOI Listing
November 2014

Anti-angiogenic effect of the basement membrane protein nidogen-1 in a mouse model of choroidal neovascularization.

Exp Eye Res 2014 Jan 23;118:80-8. Epub 2013 Nov 23.

Department of Ophthalmology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.

In patients with age-related macular degeneration disruption of the integrity of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM), precedes choroidal neovascularization (CNV). We investigated the role of the basement membrane (BM) proteins nidogen-1 and nidogen-2 for the development of experimental CNV. Laser-induced CNV was studied in Nid1(-/-) and Nid2(-/-) mice and wild type (WT) controls by fluorescein angiography, by immune histochemistry of flat-mounts or paraffin sections to analyze expression pattern of nidogen-1 and -2 and nidogen binding BM proteins, and by western blotting. The influence of VEGF and bFGF on the mRNA expression of nidogen-1 was studied in vitro. Nidogen-1 protein is present in the BM of the inner limiting membrane (ILM), the retinal capillaries, and the choroid/sclera and CNV. Nidogen-2 protein is also found in these BMs but with a weaker expression in the ILM. In the retina the absence of nidogen-1 does not influence the expression of nidogen-2 and vice versa and does not influence the expression of the BM components collagen IV, laminin γ1, and perlecan. In Nid1(-/-) mice, CNV lesions showed increased vessel leakage during angiography and the CNV area was larger than in WT or nidogen-2 deficient mice. Laser treatment led to up-regulation of nidogen-1 protein expression in the sclera/choroid of nidogen-2 deficient or WT mice. The treatment of HUVECs with VEGF leads to a reduced expression of nidogen-1 mRNA whereas its expression remained unchanged in RPE cells. In conclusion, nidogen-1 produced by the endothelial cells acts as a factor to help stabilizing the BM, thus preventing the sprouting of new vessels or the infiltration of endothelial cells. In this sense nidogen-1 is essential to provide an anti-angiogenic environment of differentiated vessels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exer.2013.11.006DOI Listing
January 2014

Skin basement membrane: the foundation of epidermal integrity--BM functions and diverse roles of bridging molecules nidogen and perlecan.

Biomed Res Int 2013 21;2013:179784. Epub 2013 Mar 21.

Department of Dermatology, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany.

The epidermis functions in skin as first defense line or barrier against environmental impacts, resting on extracellular matrix (ECM) of the dermis underneath. Both compartments are connected by the basement membrane (BM), composed of a set of distinct glycoproteins and proteoglycans. Herein we are reviewing molecular aspects of BM structure, composition, and function regarding not only (i) the dermoepidermal interface but also (ii) the resident microvasculature, primarily focusing on the per se nonscaffold forming components perlecan and nidogen-1 and nidogen-2. Depletion or functional deficiencies of any BM component are lethal at some stage of development or around birth, though BM defects vary between organs and tissues. Lethality problems were overcome by developmental stage- and skin-specific gene targeting or by cell grafting and organotypic (3D) cocultures of normal or defective cells, which allows recapitulating BM formation de novo. Thus, evidence is accumulating that BM assembly and turnover rely on mechanical properties and composition of the adjacent ECM and the dynamics of molecular assembly, including further "minor" local components, nidogens largely functioning as catalysts or molecular adaptors and perlecan as bridging stabilizer. Collectively, orchestration of BM assembly, remodeling, and the role of individual players herein are determined by the developmental, tissue-specific, or functional context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2013/179784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618921PMC
November 2013

Loss of epidermal MMP-14 expression interferes with angiogenesis but not with re-epithelialization.

Eur J Cell Biol 2012 Oct 18;91(10):748-56. Epub 2012 Jun 18.

Department of Dermatology and Venerology, University of Cologne, Germany.

Synthesis and activation of matrix metalloproteinases during wound healing are important for remodeling the extracellular matrix and modulating various cellular functions. The membrane-type 1 matrix metalloproteinase (MMP-14) has been shown to play a key role during these processes. To analyze the function of epidermal-derived MMP-14 during skin repair we generated mice lacking MMP-14 expression in the epidermis (MMP-14(ep-/-)). These mice displayed overall normal skin morphology and epidermal differentiation patterns. Wound repair in MMP-14(ep-/-) followed the same kinetics as in wild type mice (MMP-14(ep+/+)), and infiltration of neutrophils, leukocytes, and macrophages into the wound site was comparable. Microscopic analysis showed no altered re-epithelialization in the absence of epidermal MMP-14. Furthermore, epidermal differentiation at the end of the repair process and scar formation was normal. However, at day 14 post wounding, sustained angiogenesis was observed in MMP-14(ep-/-) mice in contrast to control mice. Interestingly, decreased levels of endostatin were detected in wound lysates of MMP-14(ep-/-) mice as well as in cultured keratinocytes. Taken together, these data indicate that MMP-14 expression in keratinocytes is dispensable for skin homeostasis and repair, but plays a crucial role in the epidermal-dermal crosstalk leading to modulation of vessel density.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2012.05.003DOI Listing
October 2012

Different domains in nidogen-1 and nidogen-2 drive basement membrane formation in skin organotypic cocultures.

FASEB J 2012 Sep 23;26(9):3637-48. Epub 2012 May 23.

Department of Dermatology, University Hospital of Cologne, Germany.

Nidogen-1 and nidogen-2 are homologous proteins found in all basement membranes (BMs). They show comparable binding activities in vitro and partially redundant functions in vivo. Previously, we showed that in skin organotypic cocultures, BM formation was prevented in the absence of nidogens and that either nidogen was able to rescue this failure. We now dissected the two nidogens to identify the domains required for BM deposition. For that purpose, HaCaT cells were grown on collagen matrices containing nidogen-deficient, murine fibroblasts. After addition of nidogen-1 or nidogen-2 protein fragments comprising different binding domains, BM deposition was analyzed by immunofluorescence and electron microscopy. We could demonstrate that the rod-G3 domain of nidogen-2 was sufficient to achieve deposition of BM components at the epidermal-collagen interface. In contrast, for nidogen-1, both the G2 and G3 domains were required. Immunoblot analysis confirmed that all BM components were present in comparable amounts under all culture conditions. This finding demonstrates that nidogens, although homologous proteins, exert their effect on BM assembly through different binding domains, which may in turn result in alterations of BM structure and functions, thus providing an explanation for the phenotypical differences observed between nidogen-1 and -2 deficient mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.11-194597DOI Listing
September 2012

Expression of platelet-derived growth factor-C and insulin-like growth factor I in hepatic stellate cells is inhibited by miR-29.

Lab Invest 2012 Jul 7;92(7):978-87. Epub 2012 May 7.

Institute for Pathology, University Hospital of Cologne, Koeln, Germany.

MicroRNAs are short noncoding, endogenous RNA species that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of mRNAs. Recently, it was shown that miR-29 inhibits expression of extracellular matrix proteins such as collagens, suggesting an antifibrotic function of miR-29. In the present study, we now investigated the role of miR-29 in profibrogenic growth factor expression as a further central mechanism of fibrosis. Screening of databases revealed putative miR-29 target sequences in the mRNA of platelet-derived growth factor (PDGF)-B, PDGF-B receptor, PDGF-C, vascular endothelial growth factor-A, and insulin-like growth factor (IGF)-I. To analyze miR-29 interaction with the predicted binding sites, we cloned the 3'-UTR sequences of the putative targets in fusion to the luciferase-reporter coding sequence. Functional miR-29 binding to PDGF-C and IGF-I mRNA sequences, but not to the corresponding mutants, was then proven by reporter assays. Hepatic stellate cells (HSC) that transdifferentiate into myofibroblasts, producing extracellular matrix proteins and profibrogenic growth factors, for example, the members of the PDGF family, are crucial for liver fibrosis. Myofibroblastic transition of primary HSC resulted in the loss of miR-29, but in a significant increase of PDGF-C and IGF-I. Compensation of reduced miR-29 levels by miR-29 overexpression in myofibroblastic HSC was followed by a definitive repression of IGF-I and PDGF-C synthesis. After experimental fibrosis, induced by bile-duct occlusion, miR-29 expression was shown to be reduced, but IGF-I and PDGF-C expression was upregulated, correlating inversely to the miR-29 pattern. Thus, we conclude that miR-29, downregulated during fibrosis, acts as an antifibrogenic mediator not only by targeting collagen biosynthesis, but also by interfering with profibrogenic cell communication via PDGF-C and IGF-I.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/labinvest.2012.70DOI Listing
July 2012

Epidermal transglutaminase (TGase 3) is required for proper hair development, but not the formation of the epidermal barrier.

PLoS One 2012 4;7(4):e34252. Epub 2012 Apr 4.

Center for Biochemistry, University of Cologne, Cologne, North Rhine-Westphalia, Germany.

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034252PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319564PMC
August 2012

Absence of the basement membrane component nidogen 2, but not of nidogen 1, results in increased lung metastasis in mice.

J Histochem Cytochem 2012 Apr 19;60(4):280-9. Epub 2012 Jan 19.

Department of Dermatology, University Hospital Cologne, Cologne, Germany.

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1369/0022155412436586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351237PMC
April 2012

Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction.

PLoS One 2011 9;6(9):e24568. Epub 2011 Sep 9.

Institute for Pathology, University Hospital Cologne, Cologne, Germany.

Background: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.

Methodology: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.

Principal Findings: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.

Conclusions: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024568PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170366PMC
June 2012

The disintegrin-like and cysteine-rich domains of ADAM-9 mediate interactions between melanoma cells and fibroblasts.

J Biol Chem 2011 Feb 6;286(8):6801-7. Epub 2010 Dec 6.

Department of Dermatology and Center for Molecular Medicine, University of Cologne, 50937 Cologne, Germany.

A characteristic of malignant cells is their capacity to invade their surrounding and to metastasize to distant organs. During these processes, proteolytic activities of tumor and stromal cells modify the extracellular matrix to produce a microenvironment suitable for their growth and migration. In recent years the family of ADAM proteases has been ascribed important roles in these processes. ADAM-9 is expressed in human melanoma at the tumor-stroma border where direct or indirect interactions between tumor cells and fibroblasts occur. To analyze the role of ADAM-9 for the interaction between melanoma cells and stromal fibroblasts, we produced the recombinant disintegrin-like and cysteine-rich domain of ADAM-9 (DC-9). Melanoma cells and human fibroblasts adhered to immobilized DC-9 in a Mn(2+)-dependent fashion suggesting an integrin-mediated process. Inhibition studies showed that adhesion of fibroblasts was mediated by several β1 integrin receptors independent of the RGD and ECD recognition motif. Furthermore, interaction of fibroblasts and high invasive melanoma cells with soluble recombinant DC-9 resulted in enhanced expression of MMP-1 and MMP-2. Silencing of ADAM-9 in melanoma cells significantly reduced cell adhesion to fibroblasts. Ablation of ADAM-9 in fibroblasts almost completely abolished these cellular interactions and melanoma cell invasion in vitro. In summary, these results suggest that ADAM-9 expression plays an important role in mediating cell-cell contacts between fibroblasts and melanoma cells and that these interactions contribute to proteolytic activities required during invasion of melanoma cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.168617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057808PMC
February 2011

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain.

J Biol Chem 2011 Jan 17;286(3):1911-8. Epub 2010 Nov 17.

Department of Dermatology, University Hospital of Cologne, 50937 Cologne, Germany.

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.149864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023487PMC
January 2011

Combined paracrine and endocrine AAV9 mediated expression of hepatocyte growth factor for the treatment of renal fibrosis.

Mol Ther 2010 Jul 27;18(7):1302-9. Epub 2010 Apr 27.

Institute for Pathology, University of Cologne, Cologne, Germany.

In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1alpha1 (Col1A1), platelet-derived growth factor receptor-beta (PDGFR-beta), and alpha-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2010.71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911253PMC
July 2010

Cell-matrix interactions in dermal repair and scarring.

Fibrogenesis Tissue Repair 2010 Mar 11;3. Epub 2010 Mar 11.

Department of Dermatology, University of Cologne, Cologne, Germany.

Regulation of cellular functions during dermal repair following injury is complex and critically dependent on the interaction of cells with the surrounding extracellular matrix (ECM). The ECM comprises various families of macromolecules that form the structural scaffold of the tissue, but also carry distinct biological activities. After injury to the skin, the defect is filled by a provisional matrix that is invaded by inflammatory cells, sprouting blood vessels and fibroblasts. In a later phase, the wound contracts, the tissue is replaced by mature connective tissue produced by activated fibroblasts, and a scar is formed. All cells involved communicate directly with the ECM by integrins and other matrix receptors. These transmit signals and induce adaptive responses to the environment by the embedded cells. The ECM or proteolytic fragments of individual ECM constituents exert defined biological activities influencing cell survival, differentiation of myofibroblasts, ECM synthesis and turnover, wound angiogenesis and scar remodeling. Extensive crosstalk exists between ECM and growth factors, and between growth factors and integrins. ECM-cell contact also enables direct transmission of mechanical tension, which then modulates many activities of all cellular players. Understanding this complex interplay is important to provide a basis for designing effective wound therapy and for strategic interference with mechanisms that have gone out of control in fibrotic conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1755-1536-3-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855519PMC
March 2010

Impaired wound healing in mice lacking the basement membrane protein nidogen 1.

Matrix Biol 2010 Jan 18;29(1):15-21. Epub 2009 Sep 18.

Department of Dermatology, University Hospital Cologne, Cologne, Germany.

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable alpha-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the beta1 and beta4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2009.09.004DOI Listing
January 2010

Basement membrane protein nidogen-1 shapes hippocampal synaptic plasticity and excitability.

Hippocampus 2010 May;20(5):608-20

Center for Biochemistry and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany.

The basement membrane (BM) is a specialized form of extracellular matrix (ECM) underlying epithelia and endothelia and surrounding many types of mesenchymal cells. Nidogen, along with collagen IV and laminin, is a major component of BMs. Although certain ECM proteins such as laminin or reelin influence neuronal function via interactions with cell-surface receptors such as integrins, behavioral neurological impairments due to deficits of BM components have been recognized only recently. Here, alterations in neuronal network function underlying these behavioral changes are revealed. Using nidogen-1 knockout mice, with or without additional heterozygous nidogen-2 knockout (NID1(-/-)/NID2(+/+) or NID1(-/-)/NID2(+/-)), we demonstrate that nidogen is essential for normal neuronal network excitability and plasticity. In nidogen-1 knockouts, seizurelike behavior occurs, and epileptiform spiking was seen in hippocampal in vivo EEG recordings. In vitro, hippocampal field potential recordings revealed that lack of nidogen-1, while not causing conspicuous morphological changes, led to the appearance of spontaneous and evoked epileptiform activity, significant increase of the input/output ratio of synaptically evoked responses in CA1 and dentate gyrus, as well as of paired pulse accentuation, and loss of perforant-path long-term synaptic potentiation. Nidogen-1 is thus essential for normal network excitability and plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hipo.20660DOI Listing
May 2010

Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts.

Biochem Biophys Res Commun 2009 Jul 6;385(1):55-61. Epub 2009 May 6.

Institute for Pathology, University Hospital Cologne, Kerpener Str. 62, 50924 Koeln, Germany.

Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney. The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGFbeta. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2009.05.010DOI Listing
July 2009

Basement membranes in skin: unique matrix structures with diverse functions?

Histochem Cell Biol 2009 Jul 31;132(1):1-10. Epub 2009 Mar 31.

DGZ (B 050) Rm 145, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

The view of extracellular matrix (ECM) has evolved from a merely scaffolding and space filling tissue element to an interface actively controlling cellular activities and tissue functions. A highly specialized form of ECM is the basement membrane (BM), an ubiquitous sheet-like polymeric structure composed of a set of distinct glycoproteins and proteoglycans. In this review we are largely focusing on function and assembly of BM in skin (1) at the dermo-epidermal interface and (2) in the resident micro-vasculature. The role of the non-polymeric components perlecan and particularly nidogen is exemplified by reviewing experiments based on genetic approaches and adequate experimental skin models in vivo and in vitro. While in mice total deficiency of one of these components is eventually developmentally lethal, the severity of the defects varies drastically between tissues and also the skin models recapitulating BM formation in vitro. There is accumulating evidence that this relies on the mechanical properties, the molecular composition of the BM, the adjacent ECM or connective tissue, the dynamics of molecular assembly, and 'minor' tissue-specific modifier or adapter components. Though the role of nidogen or perlecan is still remaining a controversial issue, the statements 'being essential for BM/or not' should be consequently referred to the developmental, tissue, and functional (e.g., repair) context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00418-009-0586-0DOI Listing
July 2009

Basement membranes in skin are differently affected by lack of nidogen 1 and 2.

J Invest Dermatol 2008 Sep 20;128(9):2259-67. Epub 2008 Mar 20.

Department of Dermatology, University of Cologne, Cologne, Germany.

Nidogens have been proposed to play a key role in basement membrane (BM) formation. However, recent findings using genetic approaches and organotypic coculture models demonstrated distinct tissue requirements thus changing the classical view of BM assembly. Toward this end, we have analyzed the dermo-epidermal junction and the microvasculature in skin of nidogen-deficient mice for their BM composition and structural assembly. Histology of nidogen double-null embryos at embryonic day (E)18.5 revealed overall normal skin morphology with a regularly differentiated epidermis. However, in the dermis, numerous erythrocytes had extravasated out of the microvasculature. Residual composition and ultrastructure of the dermo-epidermal BM are not altered in the absence of nidogens, demonstrating that the deposition of laminin, collagen IV, and perlecan occurs and allows cutaneous BM formation. In contrast, in capillaries, BM formation is severely impaired in the absence of nidogens, showing an irregular, patchy distribution and a dramatically reduced deposition of collagen IV, perlecan, and particularly laminin-411. Ultrastructure revealed thin fragile walls in the small blood vessels next to the epidermis, completely lacking a distinct endothelial BM. In summary, our results indicate that in skin the laminin composition of the various BMs determines whether nidogens are required for their assembly and stabilization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2008.65DOI Listing
September 2008

Nidogens-Extracellular matrix linker molecules.

Microsc Res Tech 2008 May;71(5):387-95

Center for Biochemistry and Center for Molecular Medicine, Medical Faculty, University of Cologne, D-50924 Cologne, Germany.

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jemt.20567DOI Listing
May 2008

Role of ADAM-9 disintegrin-cysteine-rich domains in human keratinocyte migration.

J Biol Chem 2007 Oct 17;282(42):30785-93. Epub 2007 Aug 17.

Department of Dermatology and Center for Molecular Medicine, Kerpener Strasse 62, University of Cologne, 50937 Cologne, Germany.

ADAM-9 belongs to a family of transmembrane, disintegrin-containing metalloproteinases involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. The aim of this study was to analyze the expression of ADAM-9 in skin and to assess the role of this proteolytic/adhesive protein in skin physiology. In normal skin, ADAM-9 expression was detected in both the epidermis and dermis and in vitro in keratinocytes and fibroblasts. Here we report that ADAM-9 functions as a cell adhesion molecule via its disintegrin-cysteine-rich domain. Using solid phase binding assays and antibody inhibition experiments, we demonstrated that the recombinant disintegrin-cysteine-rich domain of ADAM-9 specifically interacts with the beta1 integrin subunit on keratinocytes. This was corroborated by co-immunoprecipitation. In addition, engagement of integrin receptors by the disintegrin-cysteine-rich domain resulted in ERK phosphorylation and increased MMP-9 synthesis. Treatment with the ERK inhibitor PD98059 inhibited MMP-9 induction. Furthermore, the presence of the soluble disintegrin-cysteine-rich domain did not interfere with cell migration on different substrates. However, keratinocytes adhering to the immobilized disintegrin-cysteine-rich domain showed increased motility, which was partially due to the induction of MMP-9 secretion. In summary, our results indicate that the ADAM-9 adhesive domain plays a role in regulating the motility of cells by interaction with beta1 integrins and modulates MMP synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M701658200DOI Listing
October 2007

The autoantigen in anti-p200 pemphigoid is synthesized by keratinocytes and fibroblasts and is distinct from nidogen-2.

J Invest Dermatol 2008 Jan 19;128(1):87-95. Epub 2007 Jul 19.

Department of Dermatology, University Medical Center Freiburg, Freiburg, Germany.

Anti-p200 pemphigoid is a subepidermal immunobullous disorder associated with tissue-bound and circulating autoantibodies reactive with a 200 kDa protein on the dermal side of salt-split-skin. The autoantigen, named p200, is a non-collagenous glycoprotein located at the lamina lucida-lamina densa border of the epidermal basement membrane. However, its identity and cellular origin remain elusive. Here, we used biochemical and genetic approaches to characterize the autoantibody reactivity in three new patients with anti-p200 pemphigoid. We show that the target antigen p200 is synthesized by both keratinocytes and fibroblasts, is disulfide-bonded, and participates in calcium-dependent molecular interactions. Lack of collagen XVII (BP 180), collagen VII, or laminin 332 (laminin 5) from the dermal-epidermal junction does not destabilize p200. Colocalization within the basement membrane zone and an identical molecular weight suggested nidogen-2 as candidate autoantigen in anti-p200 pemphigoid, but biochemical analysis demonstrated that p200 is distinct from nidogen-2. In conclusion, the results define further the biochemical characteristics of p200 and demonstrate its in vitro-synthesis by keratinocytes and fibroblasts, thus providing a basis for identification and further characterization of this autoantigen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.jid.5700952DOI Listing
January 2008

Morphometric analysis of murine skin wound healing: standardization of experimental procedures and impact of an advanced multitissue array technique.

Wound Repair Regen 2007 Jan-Feb;15(1):105-12

Institute of Pathology, University of Cologne, Cologne, Germany.

Morphometric data based on skin wounding offer important information for the characterization of the phenotype of transgenic mouse models. The goal of this study was the comparison of technical procedures concerning wounding, processing, and evaluation of samples in different mouse strains. The multitissue array technique was used to estimate its adaptability for standardized analysis in wound healing. Skin wounds between days 1 and 14 after wounding were analyzed. The influence of mouse strain (C57BI/6 vs. FVB/N mice), sex, size of the punch biopsies, and preparation of the tissue sections was investigated on 94 mice. The parameters distance between the migration tongues (deltaMT) and surface not covered by epithelium were evaluated to describe the reepithelialization, and the distance between the adnexa was chosen to measure wound contraction. In addition, the techniques to measure the area of granulation tissue (GT) were evaluated. The data illustrate the requirement of standardized conditions for skin wound-healing experiments and demonstrate that histological preparation in serial sections is mandatory to detect slight differences in wound contraction. For the analysis of cellular composition in GT, multitissue arrays are useful tools in wound-healing studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1524-475X.2006.00191.xDOI Listing
June 2007

Loss of nidogen-1 and -2 results in syndactyly and changes in limb development.

J Biol Chem 2006 Dec 5;281(51):39620-9. Epub 2006 Oct 5.

Center for Biochemistry and Center for Molecular Medicine, Medical Faculty, University of Cologne, D-50924 Cologne, Germany.

Nidogens are two ubiquitous basement membrane proteins produced mainly by mesenchymal cells. Nidogen-mediated interactions, in particular with laminin, collagen IV, and perlecan have been considered important in the formation and maintenance of the basement membrane. However, whereas mice lacking both nidogen isoforms or carrying mutations in the high affinity nidogen-binding site upon the laminin gamma1 chain have specific basement membrane defects in certain organs, particularly in the lung, characterization of these mice has also shown that basement membrane formation per se does not need nidogens or the laminin-nidogen interaction. Limb development requires the complex interplay of numerous growth factors whose expression is dependent upon the apical ectodermal ridge. Here, we show that lack of nidogen-1 and -2 results in a specific and time-limited failure in the ectodermal basement membrane of the limb bud. The absence of this basement membrane leads to aberrant apical ectodermal ridge formation. It also causes altered distribution of growth factors, such as fibroblast growth factors and leads to a fully penetrant soft tissue syndactyly caused by the dysregulation of interdigital apoptosis. Further, in certain animals more severe changes in bone formation occur, providing evidence for the interplay between growth factors and the extracellular matrix.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M607886200DOI Listing
December 2006

Lack of nidogen-1 and -2 prevents basement membrane assembly in skin-organotypic coculture.

J Invest Dermatol 2007 Mar 28;127(3):545-54. Epub 2006 Sep 28.

Department of Dermatology, University of Cologne, Cologne, Germany.

Nidogens are considered as classical linkers joining laminin and collagen IV networks in basement membranes (BMs); however, recent genetic approaches have suggested that nidogens function in a tissue-specific and developmental context. Thus, in mice lacking both nidogen-1 and -2 heart and lung were severely affected, causing neonatal death. Furthermore, in various locations, extravasation of erythrocytes was observed implying microvascular defects. Mice expressing solely either isoform, had a functional BM, although nidogen-2 binds with lower affinity to the laminin gamma1 chain. Having previously blocked BM formation by interfering with nidogen-1 binding to laminin in skin-organotypic cocultures, here we investigated the roles of nidogen-1 and -2 in this model. For that purpose, human HaCaT cells were grown in three-dimensional cocultures on collagen matrices containing murine fibroblasts of varying nidogen deficiency. As with our experiments blocking laminin-nidogen interaction, lack of both nidogens completely prevented BM deposition and ultrastructural assembly of BM and hemidesmosomes, although other BM proteins remained detectable at comparable levels with no signs of degradation. Supplementation by recombinant nidogen-1 or -2 restored these structures, as shown by immunofluorescence and electron microscopy, confirming that in this system nidogen-2 is equivalent to nidogen-1, and both can promote the development of a functional BM zone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.jid.5700562DOI Listing
March 2007

Identification of susceptibility loci for skin disease in a murine psoriasis model.

J Immunol 2006 Oct;177(7):4612-9

Department of Dermatology and Allergic Diseases, University of Ulm, Maienweg 12, D-89081 Ulm, Germany.

Psoriasis is a frequently occurring inflammatory skin disease characterized by thickened erythematous skin that is covered with silvery scales. It is a complex genetic disease with both heritable and environmental factors contributing to onset and severity. The CD18 hypomorphic PL/J mouse reveals reduced expression of the common chain of beta(2) integrins (CD11/CD18) and spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, CD18 hypomorphic C57BL/6J mice do not demonstrate this phenotype. In this study, we have performed a genome-wide scan to identify loci involved in psoriasiform dermatitis under the condition of low CD18 expression. Backcross analysis of a segregating cross between susceptible CD18 hypomorphic PL/J mice and the resistant CD18 hypomorphic C57BL/6J strain was performed. A genome-wide linkage analysis of 94 phenotypically extreme mice of the backcross was undertaken. Thereafter, a complementary analysis of the regions of interest from the genome-wide screen was done using higher marker density and further mice. We found two loci on chromosome 10 that were significantly linked to the disease and interacted in an additive fashion in its development. In addition, a locus on chromosome 6 that promoted earlier onset of the disease was identified in the most severely affected mice. For the first time, we have identified genetic regions associated with psoriasis in a mouse model resembling human psoriasis. The identification of gene regions associated with psoriasis in this mouse model might contribute to the understanding of genetic causes of psoriasis in patients and pathological mechanisms involved in development of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.177.7.4612DOI Listing
October 2006

Nidogen and nidogen-associated basement membrane proteins and neuronal plasticity.

Neurodegener Dis 2006 ;3(1-2):56-61

Institute of Physiology, University of Rostock, Rostock, Germany.

Extracellular matrix (ECM) proteins are thought to subserve structural functions as, for example, tissue barriers as well as guidance structures during cell growth, differentiation and tissue repair. Deletion of basement membrane (BM) components results in malformations of different organs, including the brain. Recent data, however, suggest that interference with cellular membrane-associated proteins interacting with ECM can alter neuronal excitability and synaptic plasticity without obvious underlying structural damage. This does not only apply to classical ECM proteins such as laminin, reelin and tenascin, but also to molecules of a rather specialized ECM, the BM. Here, nidogen (also termed entactin) appears to subserve a function in neuronal plasticity. Nidogen ablation leads to epileptic activity in vivo and the appearance of spontaneous epileptiform activity in vitro. This raises the intriguing question whether the BM protein nidogen may directly influence neuronal function in the CNS, opening the possibility of modulatory mechanisms of synaptic plasticity and excitability reaching beyond classical processes confined to cellular interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000092094DOI Listing
October 2006

Role of the snake venom toxin jararhagin in proinflammatory pathogenesis: in vitro and in vivo gene expression analysis of the effects of the toxin.

Arch Biochem Biophys 2005 Sep;441(1):1-15

Department of Microbiology, University of Virginia, Charlottesville, VA 22908-0734, USA.

To assess the indirect effects of snake venom metalloproteinases (SVMP) on host tissue local necrosis, we investigated the effect of the SVMP jararhagin on the gene expression profiles of human fibroblasts in vitro and mouse tissue in vivo. Two functional classes of up-regulated proteins, cell death and inflammatory disease were identified as being significantly populated. The changes in gene expression observed by qRT-PCR on laser microdissected mouse muscle tissue treated with jararhagin were similar with significant up-regulation of proinflammatory transcripts such as IL-1 beta, IL-6, CXCL1, CXCL2, IL-8, and apoptosis, inflammation responsive transcripts such as TNF-alpha induced protein 6. Proteolytically inactive jararhagin had no effect on the gene expression profile of fibroblasts, indicating proteolysis as the primary mechanism affecting gene expression of cells and tissues resulting in a proinflammatory, pro-apoptotic host response which likely exacerbates the local necrosis frequently observed at the site of envenoming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.abb.2005.06.007DOI Listing
September 2005