Laminin Alpha2 Chain Research Articles

Enhanced Laminin-Derived Peptide AG73-Mediated Liposomal Gene Transfer by Bubble Liposomes and Ultrasound

A promising strategy as a cancer therapeutic is tumor-targeted gene delivery. The AG73 peptide derived from the laminin alpha1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in some cancer cells. In this study, AG73-PEG liposomes were developed for selective gene delivery to syndecan-2 overexpressing cancer cells. AG73-PEG liposomes were used in combination with Bubble liposomes and ultrasound exposure to enhance transfection efficiency by promoting the escape of the liposomes from the endosome to the cytosol. AG73-PEG liposomes showed selective gene delivery to syndecan-2 overexpressing cancer cells. Furthermore, AG73-mediated liposomal gene transfection efficiency was enhanced by 60-fold when Bubble liposomes and ultrasound exposure were used, despite the absence of an increase in the uptake of AG73-PEG liposomes into the cells. Confocal microscope analysis revealed that the Bubble liposomes and ultrasound promoted intracellular trafficking of the AG73-PEG liposomes during gene transfection. Thus, the combination of AG73-PEG liposomes with Bubble liposomes and ultrasound exposure may be a promising method to achieve selective and efficient gene delivery for cancer therapy.

The Influence of Tribenoside on Expression and Deposition of Epidermal Laminins in HaCaT Cells

Tribenoside has been used clinically for hemorrhoidal disease associated with coagulation, inflammation, and wounds. However, the pharmacological mechanism of tribenoside activity has never been clear. In this study we examined whether tribenoside affected expression and deposition of laminins that are required for reconstruction of basement membranes (BMs) during wound healing in hemorrhoidal disease. HaCaT cells, which are derived from human epidermis, were treated in growth media supplemented with tribenoside. Reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for laminin chains showed that HaCaT cells constitutively expressed laminin alpha3, alpha5, beta1, beta3, gamma1, and gamma2 chains. Tribenoside treatment of HaCaT cells did not induce expression of other laminin chains. We also quantified the expression of laminin chains in tribenoside-treated cells using real-time PCR. The expression level of laminin alpha3, beta1, beta3, gamma1, and gamma2 chains was not affected. In contrast, the expression of laminin alpha5 in the tribenoside-treated cells was four times higher than that of control cells. Immunocytochemistry also showed that tribenoside accelerated the focal deposition of laminin-332 (alpha3, beta3, gamma2). These results suggest that tribenoside interacts with epidermal cells and regulates the expression and localization of laminins to help reconstruct BMs in wound healing of hemorrhoids.

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and β1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73-induced colocalization of syndecan-1 and beta1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis.

Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin alpha chains, one beta and one Gamma, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, we mutated the gene encoding the sole laminin beta chain in Drosophila, LanB1, so that no trimers can be made. We show that LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, we show that elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, our results unravel novel functions in cell adhesion, migration and rearrangement. We propose that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution.

A Biologically Active Sequence of the Laminin α2 Large Globular 1 Domain Promotes Cell Adhesion through Syndecan-1 by Inducing Phosphorylation and Membrane Localization of Protein Kinase Cδ

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 alpha2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin alpha2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cdelta (PKCdelta) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCdelta activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 alpha2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCdelta signaling pathway.

Overexpression of malignancy‐associated laminins and laminin receptors by angiotropic human melanoma cells in a chick chorioallantoic membrane model

As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin-derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor. C8161 human melanoma cells in a shell-less chick CAM assay were used to study EVMM associated with the presence of vascularized angiotropic melanoma areas. For both high-quality histomorphology and RNA preservation in paraffin-embedded tissue, we used a methanol-based fixative coupled with microwave-assisted rapid tissue processing as previously described. Using laser capture microdissection, angiotropic melanoma areas as well as avascular areas were microdissected. Using quantitative real time polymerase chain reaction (QRT-PCR), six genes have been studied: LAMC2 (laminin gamma2 chain), LAMA4 (laminin alpha4 chain), ITGB1 (integrin beta1), ITGB3 (integrin beta3), RSPA (ribosomal protein), and MMP2 (matrix metallopeptidase 2). QRT-PCR data were normalized to human GAPDH housekeeping gene and values were compared against Human Total RNA. Final results were expressed as percentage of expression. All tumors demonstrated a similar pattern, i.e. EVMM of angiotropic melanoma cells. The microdissected histopathological sections presented both angiotropic areas and avascular areas. All genes were overexpressed in angiotropic melanoma areas vs. avascular melanoma areas, especially LAMC2, LAMA4 and ITGB3 (respectively, 165.18, 208.86, and 483.69%). This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM.

Biologically Active Sequences in the Mouse Laminin α3 Chain G Domain

The laminin alpha3 chain is mainly expressed at the skin, and its C-terminal G domain has a critical role in multiple biological functions. We screened for biologically active sites on the mouse laminin alpha3 chain G domain using 107 synthetic peptides on coated plates and conjugated to Sepharose beads with HT1080 human fibrosarcoma cells, HaCaT human skin keratinocyte cells, and human dermal fibroblasts (HDFs). Eleven peptides exhibited cell attachment activity with respect to the peptide-coated plates and/or peptide-Sepharose beads. MA3G28 (WTIQTTVDRGLL) strongly binds to HaCaT cells. Four peptides promoted PC12 cell neurite outgrowth. Heparin inhibited attachment of HDFs to eight peptides on the coated plates. In contrast, EDTA significantly inhibited attachment of HDFs to MA3G27 (NAPFPKLSWTIQ) and MA3G28 but had no effect on the attachment of the other peptides. HDF cells formed well-organized actin stress fibers and focal contacts with vinculin accumulation on MA3G27. Additionally, attachment of HDFs to MA3G27 was inhibited by anti-alpha6 and anti-beta1 integrin antibodies, suggesting that MA3G27 promotes alpha6beta1 integrin-mediated cell adhesion. MA3G57 (NQRLASFSNAQQS) exhibited cell attachment activity only in the peptide bead assay. MA3G57 conjugated to a chitosan membrane promoted HDF attachment and spreading with well-organized actin stress fibers. The anti-beta1 integrin antibody partially inhibited attachment of HDFs to the MA3G57-chitosan membrane, suggesting that the MA3G57 site is involved in beta1 integrin-mediated cell attachment. These active sites are likely important in the biological activities of the laminin alpha3 chain G domain and would be useful for the study of molecular mechanisms of laminin-receptor interactions.

Insulin-Induced Laminin Expression Promotes a Hypercontractile Airway Smooth Muscle Phenotype

Airway smooth muscle (ASM) plays a key role in the development of airway hyperresponsiveness and remodeling in asthma, which may involve maturation of ASM cells to a hypercontractile phenotype. In vitro studies have indicated that long-term exposure of bovine tracheal smooth muscle (BTSM) to insulin induces a functional hypercontractile, hypoproliferative phenotype. Similarly, the extracellular matrix protein laminin has been found to be involved in both the induction and maintenance of a contractile ASM phenotype. Using BTSM, we now investigated the role of laminins in the insulin-induced hypercontractile, hypoproliferative ASM phenotype. The results demonstrate that insulin-induced hypercontractility after 8 days of tissue culture was fully prevented by combined treatment of BTSM-strips with the laminin competing peptides Tyr-Ile-Gly-Ser-Arg (YIGSR) and Arg-Gly-Asp-Ser (RGDS). YIGSR also prevented insulin-induced increases in sm-myosin expression and abrogated the suppressive effects of prolonged insulin treatment on platelet-derived growth factor-induced DNA synthesis in cultured cells. In addition, insulin time-dependently increased laminin alpha2, beta1, and gamma1 chain protein, but not mRNA abundance in BTSM strips. Moreover, as previously found for contractile protein accumulation, signaling through PI3-kinase- and Rho kinase-dependent pathways was required for the insulin-induced increase in laminin abundance and contractility. Collectively, our results indicate a critical role for beta1-containing laminins, likely laminin-211, in the induction of a hypercontractile, hypoproliferative ASM phenotype by prolonged insulin exposure. Increased laminin production by ASM could be involved in the increased ASM contractility and contractile protein expression in asthma. Moreover, the results may be of interest for the use of inhaled insulin administrations by diabetics.

Expression of Laminin Isoforms, Receptors, and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin gamma1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin alpha6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors, and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin alpha5 chain, laminin receptors (integrin alpha3, alpha6, beta4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors, and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

LG4–5 domains of laminin‐211 binds α‐Dystroglycan to allow myotube attachment and prevent anoikis

Poly(2-hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C(2)C(12) myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA-coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases. Incorporation of merosin (laminin-211) or the short laminin globular (LG4-5) modules of the laminin-alpha2 chain C-terminus (called 2E3) that binds alpha-dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin-binding is alpha-dystroglycan. An antibody which binds alpha-dystroglycan but which does not block laminin-binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4-5 modules of laminin-211 to alpha-dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival.

Basal lamina structural alterations in human asymmetric aneurismatic aorta

Basal lamina (BL) is a crucial mechanical and functional component of blood vessels, constituting a sensor of extracellular microenvironment for endothelial cells and pericytes. Recently, an abnormality in the process of matrix microfibrillar component remodeling has been advocated as a mechanism involved in the development of aortic dilation. We focused our attention on BL composition and organization and studied some of the main components of the Extracellular Matrix such as Tenascin, Laminins, Fibronectin, type I, III and IV Collagens. We used surgical fragments from 27 patients, submitted to operation because of aortic root aneurysm and 5 normal aortic wall specimens from heart donors without any evidence for aneurysmal or atherosclerotic diseases of the aorta. Two samples of aortic wall were harvested from each patient, proximal to the sinotubular junction at the aortic convexity and concavity. Each specimen was processed both for immunohistochemical examination and molecular biology study. We compared the convexity of each aortic sample with the concavity of the same vessel, and both of them with the control samples. The synthesis of mRNA and the levels of each protein were assessed, respectively, by RT-PCR and Western Blot analysis. Immunohistochemistry elucidated the organization of BL, whose composition was revealed by molecular biology. All pathological samples showed a wall thinner than normal ones. Basal lamina of the aortic wall evidentiated important changes in the tridimensional arrangement of its major components which lost their regular arrangement in pathological specimens. Collagen I, Laminin alpha2 chain and Fibronectin amounts decreased in pathological samples, while type IV Collagen and Tenascin synthesis increased. Consistently with the common macroscopic observation that ascending aorta dilations tend to expand asymmetrically, with prevalent involvement of the vessel convexity and relative sparing of the concavity, Collagen type IV is more evident in the concavity and Tenascin in the convexity.

Chain-Specific Heparin-Binding Sequences in the Laminin α Chain LG45 Modules

Laminin alpha chains contain five tandem globular modules (LG1-5) at the C-terminus. Here, we focused on the LG45 module, which play a critical biological role via binding to heparin/heparan sulfate, and examined their chain-specific heparin-binding affinity. The relative heparin-binding affinity of recombinant laminin alpha chain LG45 proteins was as follows: alpha5 > alpha4 > alpha1 > alpha2 and alpha3. The alpha5 chain LG45 module also promoted the strongest cell attachment. We screened heparin-binding sequences using the recombinant alpha5LG45 protein and 43 synthetic peptides. Four peptides, A5G71 (GPLPSYLQFVGI) (IC(50) = 91.8 microM), A5G77 (LVLFLNHGHFVA) (IC(50) = 7.0 microM), A5G81 (AGQWHRVSVRWG) (IC(50) = 5.9 microM), and A5G94 (KMPYVSLELEMR) (IC(50) = 0.84 microM), inhibited the heparin-binding of rec-alpha5LG45. Additionally, the same four peptides exhibited dose-dependent heparin-binding activity in a solid-phase assay. We found that the alpha5 chain LG45 module contains four heparin-binding sequences, and this number is higher than that of the other LG45 modules (alpha2 and alpha3, one sequence; alpha1 and alpha4, two sequences). The data suggest that the active sequences identified from the synthetic peptide screening contribute to the heparin-binding activity of the LG45 module. Most of the heparin-binding sequences in the LG45 modules are located in the N-terminal regions of the LG4 module within the loop regions in the proteins. The data suggest that the N-terminal loop regions of the LG4 module are mainly involved in the heparin/heparan sulfate-mediated biological functions.

Temporal and Spatial Expression Pattern of Four Laminin Alpha Chains in Xenopus laevis

Temporal and Spatial Expression Pattern of Four Laminin Alpha Chains in Xenopus laevis

Conformational requirement on peptides to exert laminin's activities and search for protein segments with laminin's activities

The human laminin alpha3 chain LG4 module has biological activities of cell adhesion, heparin binding, migration, and neurite outgrowth. The authors had previously identified that the active site of this protein is in residues 1411-1429 (amino-acid sequence = KNSFMALYLSKGRLVFALG called A3G756) and that a three-amino-acid sequence KGR in A3G756 is crucial for exerting the activities. An experiment has shown that a cyclo-hEF3A peptide (a cyclic analog of A3G756) exhibits stronger activities than a linear-hEF3A peptide (a linearized peptide of the cyclo-hEF3A peptide). This experiment implies that adopting a loop conformation may be important for exerting the activities. In this study, the authors first computed the solution structures of the cyclo-hEF3A and linear-hEF3A peptides by molecular dynamics simulations. The obtained conformational ensembles consisted of a variety of conformations, which is a usual property of short peptides in solution. The ensembles involved a fraction where the peptide adopted beta-hairpins and KGR was located at the hairpin head. If there are protein segments that adopt beta-hairpins similar to those sampled from the simulation and have the KGR sequence at the hairpin head, these segments may have some activities. Then, the authors searched a database for segments satisfying these requirements and detected six functional segments. Three of them had laminin's activity, and the remaining three had activities similar to laminin's activities. Analyses on the conformational ensembles of cyclo- and linear-hEF3A peptides suggest that not only the KGR position in the hairpin but also the inter-strand packing is important for exerting laminin's activities.

Intrinsic laryngeal muscles are spared from degeneration in the dy3k/dy3k mouse model of congenital muscular dystrophy type 1A

Deficiency of laminin alpha2 chain leads to a severe form of congenital muscular dystrophy (MDC1A). Here, we analyzed whether the intrinsic laryngeal muscles (ILM) are spared in the dy(3K)/dy(3K) mouse model of complete laminin alpha2 chain absence. No muscle degeneration was evident; expression of various laminin chains was similar to that of limb muscles, and sustained integrin alpha7B expression was noted in laminin alpha2 chain-deficient ILM. We conclude that ILM are spared in MDC1A.

Skeletal Muscle-Specific Ablation of raptor, but Not of rictor, Causes Metabolic Changes and Results in Muscle Dystrophy

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.

Genetically Confirmed Patients with Merosin-deficient Congenital Muscular Dystrophy in China

We report a family and a single patient in China involved with merosin-deficient congenital muscular dystrophy (MDC1A) with typical clinical symptoms. Pathological analysis of biopsied skeletal muscle showed dystrophic changes with proliferated fibrotic tissue elements as the predominant finding. Immunohistochemical analysis demonstrated the complete absence of the laminin alpha2 chain (merosin) around muscle fibers. In patient 1, a double mutation, c.[9101_9104dupAACA:3412G>A] p.[H3035QfsX4:V1138M] was detected, whereas her parents and another sibling were heterozygous carriers. Patient 2 had a novel homozygous nonsense mutation, c.2907C>A (p.Cys969X), in exon 21. The genotype-phenotype correlation of Chinese children with novel merosin-deficient congenital muscular dystrophy is reported.

Cib2 Binds Integrin α7Bβ1D and Is Reduced in Laminin α2 Chain-deficient Muscular Dystrophy

Mutations in the gene encoding laminin alpha2 chain cause congenital muscular dystrophy type 1A. In skeletal muscle, laminin alpha2 chain binds at least two receptor complexes: the dystrophin-glycoprotein complex and integrin alpha7beta1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin alpha2 chain-deficient mouse limb muscle. One of the down-regulated genes encodes a protein called Cib2 (calcium- and integrin-binding protein 2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin alphaIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin alpha7beta1-binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle, Cib2 colocalizes with the integrin alpha7B subunit at the sarcolemma and at the neuromuscular and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium-binding protein that interacts with integrin alpha7Bbeta1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin alpha7Bbeta1D signaling in skeletal muscle.

T.O.2 Omigapil/SNT-317 prevents apoptosis and ameliorates the pathology of laminin-alpha2 deficient muscle dystrophy

Mutations in LAMA2, the gene encoding the laminin-alpha2 chain of the extracellular matrix of muscle cells, cause a rare and severe form of congenital muscular dystrophy, called MDC1A. The disease is characterized by early onset, progressive muscle degeneration and impaired regeneration. As consequence, affected children are often never able to walk and, as there is no effective treatment available, frequently die in early childhood. Genetic evidence has shown that overexpression of the apoptosis inhibitor protein BCL-2 can protect from disease-relevant pathologies in the laminin-alpha2 deficient dyW/dyW mouse, a model for MDC1A. In addition, expression of a miniaturized form of the extracellular matrix molecule agrin (mini-agrin) has been shown to be an alternative way to ameliorate disease symptoms. The efficacy of omigapil (N-(dibenz(b,f)oxepin-10-ylmethyl)-N-methyl-N-prop-2-ynylamine maleate; SNT-317, TCH346), a chemical derivative of selegiline, was tested in dyW/dyW mice by oral administration at a daily dose of 0.1 or 1 mg/kg starting at 3-weeks of age. Control animals were treated with vehicle. We show that omigapil, a well characterized inhibitor of apoptosis that targets GAPDH, ameliorated key pathology hallmarks of the dyW/dyW mouse. Specifically, oral administration of omigapil reduced apoptosis in muscle and preserved muscle histology, reduced body weight loss, mitigated skeletal deformation and improved locomotion. Moreover, omigapil increased the 50% survival time from 35 days in vehicle treated dyW/dyWmice to 85 days and 105 days in dyW/dyW mice treated with 0.1 and 1 mg/kg omigapil, respectively. In addition, we show that co-administration of mini-agrin had additive beneficial effects. The preclinical and clinical development of omigapil is well advanced and omigapil was proven to be safe in large clinical trials. Based on its efficacy in the dyW/dyW mouse, this orally bioavailable drug is well suited to be tested clinically as a potential therapy for MDC1A.

Localization of laminin α3B chain in vascular and epithelial basement membranes of normal human tissues and its down-regulation in skin cancers

The basement membrane (BM) proteins laminins, which consist of alpha, beta and gamma chains, play critical roles in the maintenance of tissue structures. One of laminin alpha chains, alpha3 has two isoforms, the truncated form alpha3A and the full-sized form alpha3B. In contrast to alpha3A laminins, little is known about alpha3B laminins. To show the histological distribution of the laminin alpha3B chain, we prepared alpha3B-specific monoclonal antibodies. Immunohistochemical analysis showed that the alpha3B chain was colocalized with the alpha3A, beta3 and gamma2 chains in the epithelial BMs of the skin, esophagus, breast and lung, suggesting the presence of laminin-3B32 (laminin-5B) and laminin-3A32 (laminin-5A). In the lung alveoli, laminin-3B32 was dominant over laminin-3A32, but vice versa in other epithelial BMs. In contrast, the BMs of blood vessels including capillaries were strongly positive for alpha3B, but almost or completely negative for alpha3A, beta3 and gamma2. alpha3B was colocalized with beta1 and gamma1 in these BMs. The alpha3B chain was scarcely detected in the vessels of malignant skin cancers, though the gamma2 and beta3 chains were highly expressed in the cancer cells. These results strongly suggest that the laminin alpha3B chain is widely expressed in vascular BMs of normal tissues, probably as laminin-3B11/3B21 (laminin-6B/7B).