Expression Of Laminin Research Articles

Disruption of Laminin-γ1 Expression Induces Rapid Decrease In Spleen Size.

AbstractAbstract 2596Laminins are heterotrimeric proteins composed of an alpha, beta, and gamma subunit, and are expressed in a majority of organ systems, including hematopoietic and lymphoid tissues. Specific laminin subunits have been identified in prognostic stromal gene expression profiles from diffuse large B-cell lymphomas (Lenz et al. 2008). Laminin g1 is the gamma-subunit present in a majority of physiologically expressed laminin heterotrimers. Global laminin-g1 deficiency in mice is embryologically lethal at E5.5 and results in part from failure of the primordial basal lamina to form (Smyth et al. 1998). Conditional knockout mouse lines have therefore been developed to examine the role of laminin-g1 in specific tissues. Disruption of Schwann cell-specific expression of laminin-g1 prevents basal lamina formation within peripheral nerves, decreases glial cell proliferation, and increases glial cell apoptosis leading to a severe dysmyelinated phenotype (Chen and Strickland 2003; Yu et al. 2005).Here we present data that global disruption of laminin- g1 expression in adult animals induces involution of the spleen and suggests a fundamental role for laminins in maintenance of spleen integrity. Mice homozygous for the LoxP flanked laminin-g1 allele (floxed allele) were crossed with mice expressing a global Cre-ERT2 transgene. Mice homozygous or heterozygous for the floxed laminin-g1 allele that carry the inducible Cre transgene are hereafter referred to as homozygous and heterozygous mutant mice, respectively. Adult heterozygous and homozygous mutant animals were treated with either tamoxifen (TM) or corn oil (control), and three weeks afterwards, tissues were harvested for analysis. Treatment of homozygous mutant mice with TM, but not corn oil, induced laminin-g1 gene recombination. There was a significant decrease in spleen size in TM-treated but not control-treated homozygous mutant animals. Neither TM nor control treatment induced changes in the spleen size of heterozygous mutant animals. Similarly, TM treatment did not alter spleen size of animals that expressed Cre-transgene but were wild type with respect to the floxed laminin-g1 gene, or which were homozygous for the floxed laminin-g1 gene but lacked the Cre-transgene (Figure 1). [Display omitted] This is the first animal model to show that continued expression of laminins are critical for maintenance of the normal spleen and suggests a potential new target for disorders of lymphoid or splenic hyperproliferation.Chen ZL, Strickland S. 2003. Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve. J Cell Biol 163(4):889-99.Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, Xu W, Tan B, Goldschmidt N, Iqbal J and others. 2008. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 359(22):2313–23.Smyth N, Vatansever HS, Meyer M, Frie C, Paulsson M, Edgar D. 1998. The targeted deletion of the LAMC1 gene. Ann N Y Acad Sci 857:283–6.Yu WM, Feltri ML, Wrabetz L, Strickland S, Chen ZL. 2005. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci 25(18):4463–72. Disclosures:No relevant conflicts of interest to declare

Glial dystrophin-associated proteins, laminin and agrin, are downregulated in the brain of mdx mouse

In this study, we investigated the involvement of dystrophin-associated proteins (DAPs) and their relationship with the perivascular basement membrane in the brains of mdx mice and controls at the age of 2 months. We analyzed (1) the expression of glial DAPs α–β-dystroglycan (DG), α-syntrophin, aquaporin-4 (AQP4) water channel, Kir 4.1 and dystrophin isoform (Dp71) by immunocytochemistry, laser confocal microscopy, immunogold electron microscopy, immunoblotting and RT-PCR; (2) the ultrastructure of the basement membrane and expression of laminin and agrin; and (3) the dual immunofluorescence colocalization of AQP4/α–β-DG, and of Kir 4.1/agrin. The following results were observed in mdx brain as compared with controls: (1) a significant reduction in protein content and mRNA expression of DAPs; (2) ultrastructurally, a thickened and discontinuous appearance of the basement membrane and a significant reduction in laminin and agrin; and (3) a molecular rearrangment of α–β-DG, coupled with a parallel loss of agrin and Kir 4.1 on basement membrane and glial endfeet. These data indicate that in mdx brain the deficiency in dystrophin and dystrophin isoform (Dp71) is coupled with a reduction of DAP components, coupled with an altered anchoring to the basement membrane.

Cytoplasmic and stromal expression of laminin γ 2 chain correlates with infiltrative invasion in ovarian mucinous neoplasms of gastro-intestinal type

Ovarian mucinous neoplasms of gastro-intestinal type (GI-type) are known to be a heterogeneous tumor composed of benign, borderline and non-invasive and invasive malignant lesions. The presence of infiltrative invasion is also known to be an important prognostic factor of this neoplasm. Laminin γ 2 chain, known to stimulate tumor cell invasion and migration, has not been sufficiently investigated in ovarian mucinous neoplasms. The purpose of this study was thus to clarify the role of laminin γ 2 in ovarian mucinous neoplasms of GI-type. We selected each morphological phase of tumor development from 61 cases of mucinous neoplasms of the GI-type: 55 adenoma lesions, 60 borderline lesions, 20 microinvasive lesions, 17 intraepithelial carcinoma lesions, 38 expansile invasive carcinoma lesions, 19 infiltrative invasive carcinoma lesions and 5 mural nodules lesions; and evaluated the localization of laminin γ 2 in the lesions using immunohistochemical method. The staining pattern was classified into i) basement membranous (BM), ii) cytoplasmic (CYT) and iii) stromal (S) pattern. The BM pattern was characteristic in adenoma, borderline, and interaepithelial and expansile invasive carcinoma lesions. The CYT and S patterns were characteristic in infiltrative invasive lesions. The staining pattern of mural nodules was similar to that of infiltrative invasion. The infiltrative invasion of GI-type ovarian mucinous neoplasms may be promoted by cytoplasmic and/or stromal expression of laminin γ 2 chain.

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

Micron-scale spatially patterned, covalently immobilized vascular endothelial growth factor on hydrogels accelerates endothelial tubulogenesis and increases cellular angiogenic responses.

Spontaneous formation of endothelial tubules was restricted to patterned micron-scale regions presenting cell adhesion ligands and angiogenic signaling protein on poly(ethylene glycol) hydrogels. Arginine-glycine-aspartic acid-serine (RGDS), an integrin ligand, and vascular endothelial growth factor (VEGF), a rate-limiting signaling protein involved in angiogenesis, were covalently bound through photopolymerization via laser scanning lithography to the surface of poly(ethylene glycol) hydrogels in patterned micron-scale regions. Endothelial cells cultured in this restricted environment underwent accelerated tubulogenesis, forming endothelial tubes within 2 days, whereas cells cultured on larger patterned areas remained spread and did not form tubules by day 2. Tubules formed in 2 days on RGDS and VEGF patterns were observed to possess lumens; however, tubule-like structures formed in 2 days on RGDS-only control patterns did not have observable lumens. Additionally, tubules that formed on restricted areas of RGDS and VEGF expressed more VEGF receptor 1, VEGF receptor 2, and ephA7 surface markers, in addition to higher expression of laminin, than cells remaining spread on wide patterned lines. This work reports spatial control and acceleration of endothelial tubule formation using biocompatible hydrogel materials to allow the formation of highly organized vascularized tissues.

Autocrine Transforming Growth Factor-β1 Activation Mediated by Integrin αVβ3 Regulates Transcriptional Expression of Laminin-332 in Madin-Darby Canine Kidney Epithelial Cells

Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

Pattern of Laminin Expression during Kidney Morphogenesis in Balb/c Mice

Basement membrane of glomerular mesangium (BMG) is one of important components which play a key role to support of the capillary loops in a renal glomerulus and completeness of BMG due to interaction of ureteric bud and metanephric mesenchyme during glomerulogenesis. As laminin contribute in extra cellular matrix and especially in basement membrane, the aim of the present study was to demonstrate the distribution of this molecule so, in this investigation specific antibody against laminin have been used in light microscopy to study development of BMG of fetal and postnatal mouse glomerular mesangium. Female inbred Balb/c mice were selected and were kept under normal condition and finding vaginal plug was assumed as day zero of pregnancy. Two pregnant mice were sacrificed by cervical dislocation in one of gestational days 13-18, respectively and their fetuses were fixed, serially sectioned and by using antibody against laminin in BMG were carried out. The same process was used for kidneys preparation at 15 postnatal days. Present data revealed that laminin showed weak reaction on day 14 of gestation. The amount of laminin increased continuously until next days of fetal life and primary of 10 days postnatal in BMG. After this period, laminin reaction did not show significant change in newborns. These data indicate that laminin appears just during the glomerulogenesis and because of continuity with vasculature which is required for Extra Cellular Matrix (ECM) and glomerular endothelial cell differentiation, laminin, is the one of major structural proteins in BMG.

Matrix metalloproteinase-9 potentiates early brain injury after subarachnoid hemorrhage

Objective: This study investigated the role of matrix metalloproteinase-9 (MMP-9) in early brain injury after subarachnoid hemorrhage (SAH).Method: Sprague–Dawley male rats (n=36) weighing between 250 and 300 g were used. SAH was produced by injecting autologous arterial blood into the pre-chiasmatic cistern. MMP-9 protein expression and activity were measured by Western blot and zymogram; laminin expression and neuronal cell in hippocampus were studied by immunohistochemistry and TUNEL staining at 24 hours after SAH in the presence or absence of a selective MMP-9 inhibitor SB-3CT.Result: MMP-9 was activated by SAH and inhibited by SB-3CT at 24 hours after SAH (p<0.01). Laminin, the substrate of MMP-9, was decreased at 24 hours after SAH, and SB-3CT prevented laminin degradation. The number of TUNEL-positive neurons in hippocampus was increased after SAH and decreased by SB-3CT (p<0.01). In addition, brain water content and neurological functional abnormalities were attenuated by SB-3CT.Conclusion: MMP-9 may be involved in early brain injury through degradation of laminin and neuronal death, and inhibition of MMP-9 may be a potential direction for brain protection after SAH.

EGF/TGFβ1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFβ1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

BAS/BSCR41 Modulation of extracellular matrix protein expression by interleukin 1 in human cardiac myofibroblasts: regulation by p38 MAP kinase

The proinflammatory cytokine interleukin 1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac...

In vivo extracellular matrix protein expression by human periodontal ligament after stimulation with orthodontic force

It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force application in vivo is not known. The aim of this present study was to evaluate the protein expression of fibronectin, laminin and vitronectin by human PDL from teeth on which orthodontic force was applied. Twenty healthy individuals were included in the study. PDL was obtained from teeth after a 3-week treatment with orthodontic force. PDL-protein samples were separated on 7.5% SDS-PAGE Western blot analysis with specific monoclonal antibodies for fibronectin, laminin and vitronectin. Bands were visualized with an enhanced chemiluminescence detection system and densitometric. Scanning of bands was carried out to compare differences in protein expression. A significant increment in fibronectin (13.9%), laminin (16.5%) and vitronectin (14.2%) expression was found in PDL from teeth treated with orthodontic force for 3 weeks in comparison with teeth in the control group. Our results support the concept that molecular changes take place by application of orthodontic forces to the PDL. Over expression of these proteins suggests that extracellular matrix (ECM) remodeling could be generated in response to mechanical stress.

Cutaneous Schwannomas in 22 Horses

Schwannomas are uncommonly recognized in horses. This study describes cutaneous schwannomas in 22 horses aged 8 to 25 years: 12 male, 7 female, and 3 of unknown sex. The horses had solitary cutaneous masses: 9 on the head, 3 on the neck, and the others on the shoulder, hip, thorax, abdomen, rump, extremities, or tail. The location of 1 tumor was unknown. The dermal tumors were well demarcated and expansile. Twelve had a multinodular pattern, whereas 10 formed a single nodule. Antoni A areas were observed in all tumors, and 10 tumors contained Antoni B areas. In Antoni A areas, the densely packed spindle-shaped neoplastic cells were arranged in short fascicles with nuclear palisading. In the hypocellular Antoni B areas, neoplastic cells were separated by abundant myxomatous stroma. Tumors commonly had hyalinization of stroma and vessel walls and ancient change. Cellular vacuolation was observed in 18 tumors. In all 22 cases, neoplastic cells were immunopositive for S100 protein. Expression of laminin and glial fibrillary acidic protein was observed in all 6 tumors evaluated by immunohistochemistry for these markers. One tumor was examined ultrastructurally: Neoplastic cells had branched cytoplasmic processes and were surrounded by an external lamina. Follow-up information was available 8 months to 10 years postexcision for 9 horses, for which surgical excision of the tumor was curative. The equine cutaneous schwannomas in this study had microscopic features like those of human schwannoma and had benign clinical behavior. Correct classification of equine cutaneous schwannoma will facilitate accurate prognosis and appropriate treatment.

Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts

In spite of significant advantages exhibiting in the applications of silicon dioxide particles in biological and medicine fields, their adverse effects still remain a big concern. Herein monodisperse spherical SiO 2 particles with diameters of 80 nm and 500 nm were used to study their interactions with human dermal fibroblasts. Both the particles were readily internalized into the fibroblasts within a short time. The 500 nm particles were taken up in a larger amount through macropinocytosis and clathrin-mediated endocytosis pathways, whereas uptake of the 80 nm SiO 2 particles was mediated corporately by macropinocytosis, clathrin-mediated and caveolae-mediated endocytosis. The particles mainly dispersed in the cytoplasm or resided within the lysosomal vesicles, but could not enter into the cell nucleus within 24 h culture in vitro. Treatment with the 80 nm SiO 2 particles caused apparently decrease of cell viability and also weakened the mitochondrial membrane potential. Further experiments demonstrated that the cell adhesion and migration were greatly affected by uptake of the SiO 2 particles regardless of their size. RT-PCR results indicated down regulation of the mRNA expression of adhesion relevant genes, i.e. fibronectin, laminin and focal adhesion kinase (FAK).

HA modulation of epidermal morphogenesis in an organotypic keratinocyte‐fibroblast co‐culture model

Hyaluronan (HA) is a major extracellular matrix component of the skin. Amongst its biological functions is the maintenance of epidermal homeostasis. The mechanisms of action, however, remain unclear. To explore the interaction of HA with the epidermis, we have looked at the effects of exogenous application of HA in an organotypic culture model containing a dermal substrate with and without fibroblast incorporation. The results demonstrate that exogenous HA enhances epidermal proliferation resulting in a thicker viable epidermis with an increase in the number and intensity of Ki67-positive basal cells; HA also improves the basement membrane assembly as evidenced by an increased expression of laminin-332 and collagen type IV but not the expression of Nidogen-1 at the epidermal-dermal junctional zone; furthermore, the development of epidermal lipid barrier structure was enhanced. These findings provide evidence to support the therapeutic use of exogenous HA for treating skin disorders with aberrant epidermal homeostasis.

Kainic acid and 3-Nitropropionic acid induced expression of laminin in vascular elements of the rat brain

Laminin is a glycoprotein component of the basement membrane and has been reported to be found in different areas of the nervous system including brain endothelial cells, Schwann cells and peripheral nerves. Although the in-vitro studies suggest that laminin plays an important role in growth and neurite extension of cultured neurons, localization of laminin in the brain has been controversial and inconsistent results have been reported. Recently, laminin immunoreactivity has been used as a marker for vascular elements in the brain. In this study, we have investigated the effect of two mechanistically different neurotoxins, kainic acid (KA), an NMDA agonist and 3-Nitropropionic acid (3-NPA), an inhibitor of mitochondrial respiration, on brain vascular elements revealed by laminin immunolabeling. We also explored whether administration of these two neurotoxic drugs correlate with the neuronal degeneration observed after neurotoxic insult by staining with Fluoro-Jade C dye. We have employed single immunolabeling to localize laminin in the brains. In KA treated rats, most of the laminin immunoreactivity is present in the piriform cortex, corpus callosum (myelinated tracts) amygdala, hippocampus, ventral thalamus and tenia tacta. In 3-NPA treated animals, laminin immunoreactivity was confined mostly to the striatum. In contrast, saline treated rats showed very little laminin immunolabeling around capillaries, arteries and in the meningeal membranes. To determine the effects of these neurotoxins on the integrity of the blood brain barrier (BBB), endothelial brain barrier antigen (EBA) immunolabeling was also performed. In addition, we performed CD11b immunolabeling to evaluate the effect of 3-NPA and KA on the activation of microglia in the brain. CD11b was dramatically increased in KA and 3-NPA treated animals. We have also combined laminin immunolabeling with Fluoro-Jade C labeling to evaluate the spatio-temporal association of degenerating neurons and the expression of laminin containing microvessels. Areas which showed intense laminin immunolabeling following KA or 3-NPA exposure correlated with those exhibiting the greatest number of degenerating neurons observed after Fluoro-Jade C staining. EBA-laminin double immunolabeling demonstrated that the expressions of laminin were predominantly localized in the areas (cortex, thalamus and hippocampus) where EBA has been either reduced or is absent. Our results from these experiments demonstrate that vascular laminin expression increases after treatment with KA or 3-NPA, suggesting the occurrence of neovascularization. Microglia may also contribute to the neurotoxic induced neovascularization and neurodegeneration.

The role of hypoxia-inducible factor-1α, aquaporin-4, and matrix metalloproteinase-9 in blood-brain barrier disruption and brain edema after traumatic brain injury

The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α), aquaporin-4 (AQP-4), and matrix metalloproteinase-9 (MMP-9) in blood-brain barrier (BBB) permeability alterations and brain edema formation in a rodent traumatic brain injury (TBI) model. The brains of adult male Sprague-Dawley rats (400-425 g) were injured using the Marmarou closed-head force impact model. Anti-AQP-4 antibody, minocycline (an inhibitor of MMP-9), or 2-methoxyestradiol (2ME2, an inhibitor of HIF-1α), was administered intravenously 30 minutes after injury. The rats were killed 24 hours after injury and their brains were examined for protein expression, BBB permeability, and brain edema. Expression of HIF-1α, AQP-4, and MMP-9 as well as expression of the vascular basal lamina protein (laminin) and tight junction proteins (zona occludens-1 and occludin) was determined by Western blotting. Blood-brain barrier disruption was assessed by FITC-dextran extravasation, and brain edema was measured by the brain water content. Significant (p < 0.05) edema and BBB extravasations were observed following TBI induction. Compared with sham-operated controls, the injured animals were found to have significantly (p < 0.05) enhanced expression of HIF-1α, AQP-4, and MMP-9, in addition to reduced amounts (p < 0.05) of laminin and tight junction proteins. Edema was significantly (p < 0.01) decreased after inhibition of AQP-4, MMP-9, or HIF-1α. While BBB permeability was significantly (p < 0.01) ameliorated after inhibition of either HIF-1α or MMP-9, it was not affected following inhibition of AQP-4. Inhibition of MMP reversed the loss of laminin (p < 0.01). Finally, while inhibition of HIF-1α significantly (p < 0.05) suppressed the expression of AQP-4 and MMP-9, such inhibition significantly (p < 0.05) increased the expression of laminin and tight junction proteins. The data support the notion that HIF-1α plays a role in brain edema formation and BBB disruption via a molecular pathway cascade involving AQP-4 and MMP-9. Pharmacological blockade of this pathway in patients with TBI may provide a novel therapeutic strategy.

Modulatory effect of interleukin-1α on expression of structural matrix proteins, MMPs and TIMPs in human cardiac myofibroblasts: Role of p38 MAP kinase

The proinflammatory cytokine interleukin-1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac myofibroblasts (CMF), or the role of p38 mitogen-activated protein kinase (MAPK). We used a focused RT-PCR microarray to investigate the effects of IL-1α on expression of 41 ECM genes in CMF cultured from different patients, and explored regulation by p38 MAPK.IL-1α (10ng/ml, 6h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin. However, it induced marked increases in expression of specific ECM proteases, including matrix metalloproteinases MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B) and MMP-10 (stromelysin-2). Conversely, IL-1α reduced mRNA and protein expression of ADAMTS1, a metalloproteinase that suppresses neovascularization. IL-1α increased expression of TIMP-1 slightly, but not TIMP-2. Data for MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and ADAMTS1 were confirmed by quantitative real-time RT-PCR. Tumor necrosis factor-alpha (TNFα), another important myocardial proinflammatory cytokine, did not alter expression of these metalloproteinases. IL-1α strongly activated the p38 MAPK pathway in human CMF. Pharmacological inhibitors of p38-α/β (SB203580) or p38-α/β/γ/δ (BIRB-0796) reduced MMP-3 and ADAMTS1 mRNA expression, but neither inhibitor affected MMP-9 levels. MMP-1 and MMP-10 expression were inhibited by BIRB-0796 but not SB203580, suggesting roles for p38-γ/δ.In summary, IL-1α induces a distinct pattern of ECM protein and protease expression in human CMF, in part regulated by distinct p38 MAPK subtypes, affirming the key role of IL-1α and CMF in post-infarction cardiac remodeling.

TGF-β1 improves cardiac performance via up-regulation of laminin receptor 37/67 in adult ventricular cardiomyocytes

TGF-beta1 plays an important role in cardiac fibrosis, apoptosis, induction of hypertrophy and contractile dysfunction. This study investigates whether TGF-beta1 plays a role in laminin receptor 37/67 (37/67 LR)-dependent regulation of cardiac performance. Therefore, isolated adult cardiomyocytes were stimulated with TGF-beta1, the expression of the 37/67 LR was determined and cell shortening was investigated on cells attached to a non-specific, serum-based attachment substrate or to specific, laminin-coated dishes. The role of the MAP kinases in TGF-beta1-dependent induction of the 37/67 LR was examined by addition of PD98059, SB202190 and SP600125. Finally, the expression of receptor mRNA was investigated in transgenic mice constitutively over-expressing TGF-beta1 and the relationship to distress score and lung wet weight-to-body weight was analysed. TGF-beta1 induced a significant increase of the 37/67 LR mRNA and protein expression. The cytokine induced p38 MAP kinase and JNK, but not ERK. Inhibition of either p38 MAP kinase or JNK attenuated the TGF-beta1-dependent increase in 37/67 LR expression. TGF-beta1 induced a loss of cell shortening in cells attached to a non-specific substrate, but not in cells on a pre-coated laminin matrix. Inhibition of JNK attenuated the protective effect of laminin receptor up-regulation on cardiac performance. Inhibition of p38 MAP kinase attenuated the depressive effect of TGF-beta1 on basal cell shortening. In transgenic mice over-expressing TGF-beta1 a strong induction of laminin receptor expression attenuated the severeness of the mice' symptoms. This study shows a new and protective role of TGF-beta1-dependent up-regulation of the 37/67 LR in cardiomyocytes in cardiac remodelling with increased laminin expression.

Hand2 Regulates Extracellular Matrix Remodeling Essential for Gut-Looping Morphogenesis in Zebrafish

Extracellular matrix (ECM) remodeling is critical for organogenesis, yet its molecular regulation is poorly understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut leftward, allowing correct placement of the liver and pancreas. To observe LPM migration at cellular resolution, we transgenically expressed EGFP under the control of the regulatory sequences of the bHLH transcription factor gene hand2. We found that laminin is distributed along the LPM/gut boundary during gut looping, and that it appears to become diminished by the migrating hand2-expressing cells. Laminin diminishment is necessary for LPM migration and is dependent on matrix metalloproteinase (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged laminin deposition at the LPM/gut boundary, leading to failed asymmetric LPM migration and gut looping. Our study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis.

Immunohistochemial evaluation of sarcoglycans and integrins in gingival epithelium of multiple myeloma patients with bisphosphonate-induced osteonecrosis of the jaw

Osteonecrosis of the jaw (ONJ) is an adverse outcome associated to bisphosphonate treatment. However, it is not known whether the ONJ lesion originates in the bone, or whether it may initiate in the oral mucosa. The aim of our study was to evaluate the pattern of basal lamina of oral mucosa after bisphosphonate administration and to analyze the structural damage of the mucosa in ONJ patients, and in subjects treated with bisphosphonates without osteonecrosis. By immunohistochemistry, we evaluated changes in basement membrane by expression of signalling proteins, laminin, and type IV collagen. All tested proteins were almost absent in basal lamina and mucosa of subjects treated with bisphosphonates without osteonecrosis, whereas in mucosa of patients with ONJ, they showed a clearly detectable pattern of the same proteins, specifically in basal lamina, but less in comparison to control samples. Moreover, in pathological mucosa, the clearly detectable staining pattern for VEGF indicated a massive neoangiogenesis. Bisphosphonates induce changes in expression of proteins also in oral mucosa. The increase of these proteins in basal lamina, and the neo-angiogenesis, concomitant with formation of the lesion, could indicate a compensative behaviour in the remodelling of the gingival mucosa in order to restore the epithelial architecture.