Major Component Of Basement Membrane Research Articles

Abstract 18325: Functional Relationship of the COL4A1/COL4A2 Locus on Chromosome 13q34 to Coronary Artery Disease (CAD)

Background: The COL4A1/COL4A2 locus has been linked to CAD in recent large GWAS meta-analyses. The COL4A1 and COL4A2 proteins constitute the major structural component of basement membranes, have important functional roles in angiogenesis, and mutations have been associated with diverse vascular abnormalities. We performed epistasis analysis for CAD-associated SNPs at the COL4A1/COL4A2 locus; this revealed genetic interaction with members of the TGFβ/Smad signalling pathway. Furthermore, pathway analysis of GWAS has implicated TGFβ and Smad signaling as well as the extracellular matrix as enriched CAD pathways. Bioinformatic analysis also predicted binding of Smad3 and Smad4 to the promoter region of COL4A1/COL4A2 . We sought to address the hypothesis that the TGFβ-mediated regulation of COL4A1 and COL4A2 is via Smad3/Smad4 and that this pathway is relevant to the pathogenesis of CAD. Methods and Results: Under basal conditions, HT-1080 fibroblast cells treated with TGFβ1 for 16h produced 5 fold and 3 fold increases in COL4A1 and COL4A2 mRNA respectively as measured by qRT-PCR. In contrast, 16h treatment with TGFβ1 in the presence of the specific Smad pathway inhibitor SB431542 (10nM) abolished COL4A1/COL4A2 upregulation (p<0.001). To rule out the possibility that TGFβ1 regulates COL4A1 and COL4A2 expression indirectly via a non-Smad pathway, we showed that inhibition of the JNK, p38 MAPK, and ERK pathways had no effect on COL4A1 and COL4A2 levels basally or in response to TGFβ1. siRNA knockdown (>85%) of SMAD3 in HT-1080 cells diminished the TGFβ1 upregulation of both COL4A1 and COL4A2 mRNA levels in comparison to nontarget siRNA (p<0.001). The effect of TGFβ1 on COL4A1 and COL4A2 mRNA expression was completely abolished when SMAD3 and SMAD4 were knocked-down in concert. These effects on COL4A1 and COL4A2 mRNA expression were recapitulated at the protein level by Western blotting. Conclusions: These data suggest that Smad3 and Smad4 are the downstream mediators by which TGFβ1 upregulates COL4A1 and COL4A2 expression in HT-1080 cells. This work further highlights the importance of the extracellular matrix and TGFβ/Smad signalling in the pathogenesis of CAD.

A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.

The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Evaluation of the effectiveness of diabetic nephropathy stage III in patients with type 2 diabetes mellitus therapy with sulodexide

Introduction. Diabetes mellitus (DM) - takes the main place in the structure of endocrine diseases, and the third after cardiovascular and cancer pathology. In Ukraine 1.2 million of people suffer from diabetes and type 2 diabetes occurs in 85-90% of them. In 2004, 3.4 million of people died from diabetes complications. Diabetic nephropathy (DN) is a serious chronic complication of diabetes that leads to the formation of nodular or diffuse glomerulosclerosis. It is the most frequent cause of terminal chronic renal failure (CRF) in the world, accounting for over 25% of all cases of CRF. The generally accepted classification is Moggensen`s classification (1983, WHO), according to which five stages of diabetic nephropathy are identified, the first two stages of them are preclinical. Leading role in the pathogenesis of DN takes hyperglycemia, which is implemented by the phenomenon of glucosetoxicity. A lot of facts underscore the importance of inflammatory mechanisms triggered by cytokines. There's immune and non-immune theory of DN. The basis of non-immune theory is a violation of the synthesis of glycosaminoglycans (GAGs) that are a major component of the glomerular basement membrane (GMB). GAGs create its negative charge, which prevents the passage through the renal filter small negatively charged molecules, including albumin. In hemodynamic regulation leading role belongs to the renin-angiotensin-aldosterone system (RAAS). The blockade of the RAAS system with ACE inhibitors is the basis of a treatment strategy of DN. All mentioned above became a reason for study of the possibility of a new direction in the treatment of DN using the drug sulodexid, which is a natural mixture of GAGs.Objective of the research. Evaluating the effectiveness of sulodexide therapy of DN stage III in patients with type 2 diabetes.Materials and methods. The patients were divided into 2 groups. Group I consisted of 24 patients aged 40 to 68 years. 5 of them were women and 19 were men. In 20 patients from the group alimentary-constitutional obesity was noted. II group consisted of 11 people: 4 women and 7 men, aged 48 to 68 years, 6 of which were suffering from alimentary-constitutional obesity. Patients received standard treatment of type 2 diabetes and hypertension. In group I, sulodexide (600 LPL units intramuscularly 1 time per day for 10 days) was further included. The research of microalbuminuria (MAU) was conducted on admission and on the 10th day of treatment.Results. The initial values of MAU in 1st group were 92,7 ± 25,2 mcg/min. In the dynamics of monitoring rates decreased to 44,4 ± 9,6 mcg/min. The degree of decrease was 47% (p <0.05). In 2nd Group MAU initial values were 46,7 ± 23,4 mcg/min. In the dynamics of monitoring MAU decreased to 29,5 ± 12,6 mcg/min. Degree of decrease was 27% (p <0.05)Conclusions. 1) In patients with stage III DN with diabetes type 2 by including in the basic therapy of sulodexide level MAU was reliable decreased on 47%, which is on 20% lower than in the comparison group (p <0.05).2) During the research reliable direct dependence of decreased MAU rates with its initial values and degree of obesity was found. It indicates the increase in the effectiveness of the drug at a higher initial level of MAU in people with overweight.

Integrin α 6 β 1 Is the Main Receptor for Vascular Laminins and Plays a Role in Platelet Adhesion, Activation, and Arterial Thrombosis

Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α1β1γ1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α2, α4, or α5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. Using an in vitro flow assay, we show that laminin-411 (α4β1γ1), laminin-511 (α5β1γ1), and laminin-521 (α5β2γ1), but not laminin-211 (α2β1γ1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α6β1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Laminin-111-derived peptide-hyaluronate hydrogels as a synthetic basement membrane

We have identified a number of cell-adhesive peptides from laminins, a major component of basement membranes. Cell-adhesive peptides derived from basement membrane proteins are potential candidates for incorporating cell-binding activities into scaffold materials for tissue engineering. Our goal is development of a chemically synthetic basement membrane using laminin-derived cell-adhesive peptides and polymeric materials. In this study, we used hyaluronic acid (HA) as a scaffold material and laminin-derived cell-adhesive peptides, A99 (AGTFALRGDNPQG, binds to integrin αvβ3), AG73 (RKRLQVQLSIRT, binds to syndecans), and an A99/AG73 mixture (molar ratio = 9:1) conjugated to two-dimensional (2D) HA matrices. As a result, it was found that the 2D A99/AG73-HA matrices have strong biological functions, such as cell attachment, cell spreading, and neurite outgrowth, similar to that of basement membrane extract (BME)-coated plates. Next, we developed three-dimensional (3D) peptide-HA matrices using the A99/AG73 mixture. The 3D A99/AG73-HA matrices promoted cell spreading and improved cell viability and collagen gene expression. Further, PC12 neurite extension was observed in the 3D A99/AG73-HA matrices. These biological activities of the 3D A99/AG73-HA matrices were similar to those of the 3D BME matrices. These results suggest that the peptide-HA matrices are useful as 2D and 3D matrices and can be applied for tissue engineering as a synthetic basement membrane.

Immunolocalization of Intermediate Filaments and Laminin in the Oviduct of the Immature and Mature Japanese Quail (Coturnix coturnix japonica)

This study describes the distribution of vimentin, desmin, smooth muscle actin (SMA) and laminin in the oviduct of the immature and mature Japanese quail. The cytoskeletal proteins vimentin, desmin and SMA have been shown to be involved in cellular support, differentiation, migration and contractility. Laminin is a major component of basement membranes. Luminal epithelia in the infundibular and magnal regions of immature and mature birds exhibited strong vimentin immunoreactivity. Luminal epithelial cells exhibiting strong vimentin immunoreactivity were present in the isthmus and shell gland regions of only mature quails. Infundibular glandular grooves displayed strong vimentin immunostaining. In contrast, the glandular epithelia of the magnum, isthmus and shell gland were vimentin immunonegative. Fibroblasts and vascular endothelial cells in the lamina propria of the oviductal regions studied exhibited strong vimentin immunostaining. Smooth muscle cells forming the tunica muscularis and vascular tunica media displayed strong desmin and SMA immunostaining. Strong laminin immunostaining was demonstrated in the basement membranes associated with smooth muscle cells, as well as in the basement membranes underlying the luminal and glandular epithelia. In conclusion, this study has shown that the immunolocalization of desmin, SMA and laminin in the oviduct of the Japanese quail is similar to that in the domestic fowl. However, differences in the immunoexpression of vimentin in the LE of the two avian species were shown to exist. In addition, the study has shown that the immunolocalization of vimentin in the Japanese quail varies depending on the oviductal region, as well as the developmental stage of the oviduct.

Phosphorylation of RNF41 E3 ubiquitin ligase by Par-1 protein kinase required for epithelial cell polarity

The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell-cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell-ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell-ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell-ECM contacts required for epithelial cells to establish apical-basal cell polarity.

Reduction of Type IV Collagen by Upregulated miR-29 in Normal Elderly Mouse and klotho-Deficient, Senescence-Model Mouse

MicroRNA (miRNA), a small non-coding RNA that functions as a mediator in gene silencing, plays important roles in gene regulation in various vital functions and activities. Here we show that the miR-29 members are upregulated in klotho-deficient [klotho(−/−)] mice, a senescence-model animal, and also in normal elderly ICR mice relative to wild-type littermates and young ICR mice. In addition, levels of type IV collagen, a major component of basement membranes and a putative target of miR-29, were lower in klotho(−/−) and elderly ICR mice than in wild-type littermates and young ICR mice. RNA degradation mediated by miR-29 may participate in the suppression of type IV collagen, both in vivo and in vitro. Taken together, our current findings suggest that the miR-29 upregulated in aging may be involved in the downregulation of type IV collagen, leading to a possible weakening of the basal membrane in senescent tissues, and miR-29 may be a useful molecular marker of senescence.

Progression of non-small cell lung cancer: Diagnostic and prognostic utility of matrix metalloproteinase-2, C-reactive protein and serum amyloid A

Matrix metalloproteinase-2 (MMP-2) is known to degrade type IV collagen, which is a major component of the cellular basement membrane, and to be involved in the invasion and metastasis of cancer cells. On the other hand, C-reactive protein (CRP) and serum amyloid A (SAA) are acute inflammatory biomarkers that increase in various conditions including infection, inflammation, malignancy and tissue disturbance. In the present study, we examined the serum levels of MMP-2, CRP and SAA in patients with localized and metastatic non-small cell lung cancer (NSCLC) to establish the clinical significance and changes in these biomarkers during NSCLC progression. In this study, 24 NSCLC patients were diagnosed at the Kitasato University Hospital and compared with 13 healthy controls. Measurement of MMP-2 levels in serum was determined by measuring pro-MMP-2 using a one-step sandwich enzyme immunoassay. CRP and SAA levels in the serum were measured by latex nephelometry. The serum levels of MMP-2, CRP and SAA in metastatic NSCLC patients were significantly higher than in localized NSCLC patients (p<0.01). There was a significant positive correlation between serum MMP-2 and CRP levels as well as SAA levels in metastatic NSCLC patients (p<0.01). Therefore, quantitation of MMP-2, CRP and SAA in NSCLC patients may be an auxiliary indicator to monitor tumor progression and poor prognosis of NSCLC disease.

NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

Author SummaryA variety of diseases, both inherited and acquired, affect muscle tissues in humans. Critical to muscle homeostasis is the anchoring of muscle fibers to their surrounding microenvironment through cell adhesion complexes that help to resist the repeated stress experienced during muscle contraction. Genetic mutations in these complexes weaken this mechanical attachment, making fibers more susceptible to damage and death. The resulting increased fiber degeneration can eventually lead to progressive muscle-wasting diseases, known collectively as muscular dystrophies. Although clinical trials are ongoing, there is presently no way to cure the loss of muscle structure and function associated with these diseases. We identified a novel cell adhesion pathway involving integrin alpha6 that promotes adhesion of muscle cells to their microenvironment. Here, we show that activation of this pathway not only significantly reduces muscle degeneration but also improves the swimming ability of dystrophic zebrafish. We explore the likely benefits and limitations of this pathway in treating symptoms of congenital muscular dystrophies. Our findings suggest that activation of this pathway (for example, by boosting levels of NAD+) has the potential to ameliorate loss of muscle structure and function in multiple muscular dystrophies.

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: Implications for regulation of BMP signalling

The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm.

421 Functional Relationship of the COL4A1/COL4A2 Locus on Chromosome 13q34 to Coronary Artery Disease (CAD)

The COL4A1 and COL4A2 genes on chromosome 13 have been identified as new loci associated with CAD (>22,000 CAD cases & >64,000 controls) that overall discovered 13 novel regions associated with CAD. The index SNP at the COL4A1/COL4A2 locus from the CARDIoGRAM study (rs4773144) has a minor allele frequency of 0.4 and is associated with an increased risk of CAD (allele specific odds ratio=1.21 in the Ottawa Heart Study (OHS)). The goal of the current project is to elucidate how variants in the COL4A1 and COL4A2 genes associated with CAD functionally and mechanistically contribute to the CAD phenotype. Type IV collagen triple helices constitute the major structural component of basement membranes, consisting primarily of 2 COL4A1 chains arranged with 1 COL4A2 chain. COL4A1 and COL4A2 also have important functional roles in angiogenesis, and mutations are associated with diverse vascular abnormalities. In a search for functional genetic variants, we resequenced the bidirectional COL4A1/COL4A2 promoter in 500 CAD cases and 500 controls and identified four novel SNPs, in promoter/enhancer regions essential for COL4A1 and/or COL4A2 gene expression and in strong linkage disequilibrium with several OHS risk SNPs. Three of these novel SNPs are in the region necessary for COL4A2 transcription and in promoter luciferase assays with HT-1080 cells result in 15-20% decreases in COL4A2 promoter activity (p<0.005). Furthermore, a follow-up study to CARDIoGRAM with more cases and controls has identified an intronic SNP in COL4A2 with an even higher CAD association than rs4773144. 2 kb of this intronic sequence containing the CAD-associated SNP was cloned into the pGL3-Promoter vector (Promega), in which insertion of functional enhancers leads to upregulation of luciferase expression in vitro. Luciferase assays in HT-1080 cells reveal this intronic sequence acts as an enhancer due to its insertion upregulating pGL3-Promoter activity over threefold relative to controls. Future work entails narrowing down what SNP(s) in this enhancer region has actual functional consequences (ie. disrupting transcription factor binding sites). We also plan on conducting chromosome conformation capture experiments to determine whether this enhancer acts locally on COL4A1/COL4A2 or acts long-range on other genomic targets. These findings are important because misregulation of COL4A1 and COL4A2 could have important consequences relevant to CAD, including effects on basement membrane integrity and angiogenesis.

Differential expression of laminin isoforms in diabetic nephropathy and other renal diseases

AbstractLaminin a non-collagenous glycoprotein is a major component of the renal glomerular basement membrane and mesangium. Thus far eleven distinct chains have been described, permutations of which make up 15 laminin isoforms. Laminin molecules interact with cells and other matrix molecules during organ development and differentiation. We studied the distribution of laminin isoforms in patients with type 1 diabetic nephropathy, membranous nephropathy, membranoproliferative glomerulonephritis and IgA nephropathy/ Henoch–Schönlein purpura. Immunofluorescence microscopic studies with laminin-chain-specific antibodies to the α1, α2, α5, β1, β2 and γ1 chains detected α2, β1 and γ1 chain expression in the normal mesangium and α5, β2 and γ1 in normal glomerular basement membrane. Significantly, constituents of the glomerular basement membrane, α5, β2 and γ1 chains were overexpressed in kidneys with diabetic nephropathy. Initially the constituents of the mesangium increased commensurate with the degree of mesangial expansion and degree of diabetic nephropathy. Reduction in α2 chain intensity was observed with severe mesangial expansion and in the areas of nodular glomerulosclerosis. In addition, with late disease aberrant expression of α2 and β2 chains was observed in the mesangium. Glomerular basement membrane in renal disease overexpressed molecules normally present in that location. In summary, the alterations in basement membrane composition in various renal diseases seem to not only reflect the balance between synthesis and degradation of normal basement membrane constituents, but also their aberrant expression.

Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain

Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. Animals received either whole-brain irradiation (a single dose of 10 Gy γ-rays or a fractionated dose of 40 Gy γ-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR). The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.

Matrix metalloproteinase-2 functional promoter polymorphism G1575A is associated with elevated circulatory MMP-2 levels and increased risk of cardiovascular disease in systemic lupus erythematosus patients

Matrix metalloproteinase-2 (MMP-2) is a zinc dependent endonuclease that degrades type IV collagen, the major structural component of basement membranes. MMP-2 functional promoter polymorphism G1575A affects circulating level of MMP-2 and may be considered an important genetic determinant of cardiovascular disease (CVD) in systemic lupus erythematosus (SLE) patients. In this study, association between MMP-2 1575A allele with serum MMP-2, neopterin and lipid-lipoprotein levels and with SLE and developing CVD was investigated. The present case-control study consisted of 109 SLE patients with and without CVD (mean age, 35.6 years) and 101 gender- and age-matched, unrelated, healthy controls (mean age, 37.1 years) from the population in the west of Iran. MMP-2 1575G/A polymorphism was detected by polymerase chain reaction (restriction fragment length polymorphism) PCR-RFLP, serum MMP-2, neopterin and lipid levels were determined by enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and enzyme assay, respectively. The presence of MMP-2 G1575A allele was found to be associated with SLE and developed CVD (OR = 1.78, p = 0.029 and OR = 3.43, p = 0.025, respectively). The SLE patients with MMP-2 A (G/A + A/A) allele had higher MMP-2 activity (301 ± 166 vs. 194 ± 35.5, p = 0.002), neopterin (29.4 ± 39.4 vs. 7.3 ± 4.6, p = 0.005), LDL-C (120 ± 25.7 vs. 87 ± 39.3, p = 0.045) and lower HDL-C (39.6 ± 11 vs. 45.9 ± 11.8, p = 0.031) levels than the control subjects. There was a significantly positive correlation between MMP-2 level with neopterin, total cholesterol and TG levels and negative correlation with HDL-C level in SLE patients with CVD. MMP-2 G1575A allele may be a risk factor for SLE. The carriers of this allele have high levels of MMP-2, neopterin, total cholesterol and TG and lower levels of HDL, thus, they are more likely to develop heart disease.

Absence of the Basement Membrane Component Nidogen 2, but Not of Nidogen 1, Results in Increased Lung Metastasis in Mice

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.

COL4A1 Mutation: Expansion of the Phenotype

Background: COL4A1 is a major basement membrane component, providing basement membrane stability. Mutations in COL4A1 lead to a small-vessel brain disease, sometimes associated with renal disorders, cataract and tortuosity of the retinal vessels. COL4A1-related cerebral stroke is considered to result from thrombosis preferring deep venous structures in the brain.Results: Mutations in COL4A1 were diagnosed in seven infants, antenatally in one, who presented at 20 weeks of gestation with an intraventricular and a cerebellar haemorrhage; postnatally in the other six, either in the perinatal period (n=3) or in infancy (n=3). Unilateral porencephaly (PC) was present in four and was recognised at birth in three and at seven months in one. Two were preterm born sibs, showing a PC with resolving clots in the lateral ventricles. The third one with PC, presented at seven months with familiar hemiplegia. More severe cerebral stroke of antenatal onset was seen in two infants, with ventriculomegaly at 26 and 37 weeks gestation respectively. They had severe supratentorial as well as infratentorial lesions with severe cerebellar atrophy. Outcome was poor, with early death in two, severe cerebral palsy in three and moderate hemiplegia in one. The pregnancy with the affected child was still ongoing. In two infants the mutation in COL4A1 was inherited, in one the mother had a somatic mosaicism, the others had a de novo mutation of the COL4A1-gene, affecting different exons.Conclusion: COL4A1 mutation should be considered in the fetus with antenatal haemorrhage, newborns with PC and children presenting with (familiar) hemiplegia.

An Antibody to the Lutheran Glycoprotein (Lu) Recognizing the LU4 Blood Type Variant Inhibits Cell Adhesion to Laminin α5

BackgroundThe Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM.Methodology/Principal FindingsIn this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg175, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5.Conclusion/SignificanceThis function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

Laminin-332 is a major component of the dermo-epidermal skin basement membrane and maintains skin integrity. The transduction of mechanical force into electrical signals by sensory endings in the skin requires mechanosensitive channels. We found that mouse epidermal keratinocytes produce a matrix that is inhibitory for sensory mechanotransduction and that the active molecular component is laminin-332. Substrate-bound laminin-332 specifically suppressed one type of mechanosensitive current (rapidly adapting) independently of integrin-receptor activation. This mechanotransduction suppression could be exerted locally and was mediated by preventing the formation of protein tethers necessary for current activation. We also found that laminin-332 could locally control sensory axon branching behavior. Loss of laminin-332 in humans led to increased sensory terminal branching and may lead to a de-repression of mechanosensitive currents. These previously unknown functions for this matrix molecule may explain some of the extreme pain experienced by individuals with epidermolysis bullosa who are deficient in laminin-332.