Laminin Α2 Chain Research Articles

AG73-modified Bubble liposomes for targeted ultrasound imaging of tumor neovasculature

Ultrasound imaging is a widely used imaging technique. The use of contrast agents has become an indispensible part of clinical ultrasound imaging, and molecular imaging via ultrasound has recently attracted significant attention. We recently reported that “Bubble liposomes” (BLs) encapsulating US imaging gas liposomes were suitable for ultrasound imaging and gene delivery. The 12 amino acid AG73 peptide derived from the laminin α1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in blood vessels. In this study, we prepared AG73 peptide-modified BLs (AG73-BLs) and assessed their specific attachment and ultrasound imaging ability for blood vessels in vitro and in vivo. First, we assessed the specific attachment of AG73-BLs in vitro, using flow cytometry and microscopy. AG73-BLs showed specific attachment compared with non-labeled or control peptide-modified BLs. Next, we examined ultrasound imaging in tumor-bearing mice. When BLs were administered, contrast imaging of AG73-BLs was sustainable for up to 4 min, while contrast imaging of non-labeled BLs was not observed. Thus, it is suggested that AG73-BLs may become useful ultrasound contrast agents in the clinic for diagnosis based on ultrasound imaging.

Structural Requirement of Fibrogenic Laminin-Derived Peptide A119 (LSNIDYILIKAS) for Amyloid-like Fibril Formation and Cellular Activity

A119 peptide (LSNIDYILIKAS), derived from the mouse laminin α1 chain sequence (residues 1321-1332), promotes cell attachment, neurite outgrowth, and amyloid-like fibril formation. In this study, we evaluated the structural requirements of A119 for biological activities and amyloid-like fibril formation. The attachment of the cell to A119 was inhibited by heparin, and using syndecan- and glypican-overexpressed cells, it was determined that A119 specifically binds to syndecans. We also evaluated the critical residues for A119 activities using a set of alanine-substituted peptides. Cell attachment activity was significantly reduced in the Leu(1)-, Ser(2)-, Asn(3)-, Ile(4)-, Ile(7)-, Ile(9)-, and Lys(10)-substituted alanine peptides. Residues Ile(4), Ile(7), Ile(9), and Lys(10) were important for neurite outgrowth activity. Congo red staining and electron microscopic examination revealed that the Ile(4), Ile(7), Ile(9), and Ser(12) residues of A119 were required for amyloid-like fibril formation. These data suggest that the Ile residues are critical for the amyloid-like fibril formation, cell attachment, and neurite outgrowth activity of A119. Furthermore, an enantiomer of A119 showed similar amyloid-like fibril formation and increased levels of cell attachment and FAK signal transduction. These findings shed light on the mechanism of amyloid-like fibril formation and demonstrate a relationship between the ability to form amyloid-like fibrils and cell behavior.

G.P.7 Effects of overexpression of LARGE on a mouse model of congenital muscular dystrophy

Dystroglycan (DG) is a central component of dystrophin–glycoprotein complex that links extracellular matrix and cytoskeleton. α-Dystroglycanopathy is a disorder characterized by muscular dystrophy often associated with brain anomaly and eye abnormalities. Mutations of known or putative glycosyltransferases, POMGnT1, POMT1, POMT2, fukutin, FKRP and LARGE, were identified and defective glycosylation of α-DG was implicated in the pathogenesis of this disorder. Interestingly, it has been reported that overexpression of LARGE strongly increase a function of α-DG and bypasses the defective glycosylation of α-DG in cultured cells deficient in fukutin or POMGnT1. Most recently, it has been demonstrated that LARGE acts on α-DG as a bifunctional glycosyltransferase which produces repeating units of xylose and glucuronic acid. In the previous study, we have generated transgenic mice that overexpresses LARGE (LARGE Tg mice) and characterized them. LARGE Tg mice were born according to the Mendelian ratio and grew normally. The mice exhibited no obvious abnormal behavior. In the mice, α-DG was hyperglycosylated and its laminin binding activity was greatly increased ubiquitously. Because the enhanced binding of α-DG with laminin should increase the stability of sarcolemma and facilitate signal transductions through α-DG, it is conceivable that the overexpression of LARGE could ameliorate the degeneration of muscle cells even in other types of muscular dystrophy than α-dystroglycanopathy. In this study, we crossed LARGE Tg mice with dy2J mice, one of a model mouse of congenital muscular dystrophy 1A in which laminin α2 chain is deficient. The dy2J mice overexpressing LARGE (dy2J/LARGE mice) have been born and we are currently characterizing them. In this meeting, we will present the detailed phenotype of dy2J/LARGE mice particularly focusing on its muscle pathology.

G.P.13 Merosin-deficient congenital muscular dystrophy: A case with immunohistochemical analysis

Congenital muscular dystrophies (CMDs) are an autosomal recessive and heterogeneous diseases. The CMDs are subclassified into two major categories: merosin positive and deficient. Primary merosin (laminin α2 chain)-deficient congenital muscular dystrophy (CMD) is a uncommon and severe form of CMD, which is caused by the mutations in the laminin α2 chain gene. In European studies reported to approximately a half of CMDs are merosin-deficient congenital muscular dystrophy (MDCMD), but it is rarely reported in asian. There were few case reports of MDCMD in Korea, we report a case of MDCMD confirmed by the immunohistochemical analysis of the frozen muscle biopsy. A 7-months-year old female was evaluated for floppy infant syndrome. She was born healthy at IUP 38weeks without perinatal distress. She had neonatal hypotonia, a high serum creatine kinase level of 3130IU/L. Her brain MRI showed a diffuse white matter change on T2WI. There was no family history of muscle weakness or developmental delay. The muscle biopsy showed a marked dystrophic pattern with many endomysial inflammatory cell infiltration. Immunohistochemical staining for dystrophin-C, α-sarcoglycan, β-sarcoglycan and α-dystroglycan proteins were decreased, and for dystrophin-R, dystrophin-N, γ-sarcoglycan, δ-sarcoglycan, dysferlin, caveolin-3 proteins were normal. We report a case of merosin-deficiency congenital muscular dystrophy confirmed by muscle biopsy.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.

Comparative Analysis of the Basement Membrane Composition of the Human Limbus Epithelium and Amniotic Membrane Epithelium

Human amniotic membrane has been widely used as substrate for ex vivo expansion and transplantation of limbal epithelial cells. To further clarify its suitability as a surrogate niche for limbal stem cells and progenitor cells, we analyzed the composition of the amniotic epithelial basement membrane, with special focus on the expression of limbus-specific matrix components. Cryosections of corneoscleral specimens obtained from 10 human donor eyes and of 6 amniotic membrane specimens obtained at cesarean section were stained by indirect immunofluorescence using a broad panel of antibodies against basement membrane components. Both amniotic and limbal epithelial basement membranes showed positive immunoreactivity for collagen type IV α1, α2, α5, and α6 chains; collagens type VII, XV, XVI, XVII, and XVIII; laminin α3, β1, β2, β3, γ1, and γ2 chains; laminin-111 and laminin-332; nidogen-1 and nidogen-2; fibronectin; fibulin-2; fibrillin-2; perlecan; and agrin. Both types of basement membrane were negative for collagen type IV α3 and α4 chains, collagen type V, and laminin α4 chain. Limbal basement membrane components, which were not detected in amniotic membrane, included laminin α1, α2, α5, and γ3 chains; BM40/SPARC; tenascin-C; matrilin-2; endostatin; and collagen type XVIII. Despite extensive similarities in basement membrane composition between amniotic and corneolimbal epithelia, the lack of limbus-specific environmental factors argues against the potential of denuded amniotic membrane as a surrogate niche for limbal stem cells but supports its suitability as a substrate to promote the formation of a well-differentiated stratified corneal epithelial equivalent for tissue engineering strategies.

Screening of integrin-binding peptides from the laminin α4 and α5 chain G domain peptide library

Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin α4 and α5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin β1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3β1- and α6β1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.

The potential of laminin-2-biomimetic short peptide to promote cell adhesion, spreading and migration by inducing membrane recruitment and phosphorylation of PKCδ

Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand–receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3β1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3β1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/β inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3β1 integrin, and that binding of α3β1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.

Improvement of Neural Stem Cell Survival in Collagen Hydrogels by Incorporating Laminin-Derived Cell Adhesive Polypeptides

Cell transplantation is a potential methodology for the treatment of Parkinson's disease. However, the therapeutic effect is limited by poor viability of transplanted cells. To overcome this problem, we hypothesized that a dual step approach, whereby providing an adhesive substrate for transplanted cells and, at the same time, by preventing the infiltration of activated microglia into the site of transplantation promotes the cell survival. To establish above conditions, attempts were made to prepare 3-D matrices using collagen hydrogels that incorporated integrin-binding polypeptides derived from laminin-1. Tandem combinations of laminin globular domains as well as a single globular domain 3 were prepared using recombinant DNA technology as a fusion with hexahistidine and bound to metal chelated surfaces to screen for the adhesion and proliferation of neural stem cells (NSCs). In addition, a small peptide derived from laminin γ1 chain was prepared and heterodimerized with the globular domain-containing chimeric proteins to evaluate for the enhancement of integrin-mediated cell adhesion. As a result, a heterodimer consisting of the globular domain 3 of the laminin α1 chain and the peptide from the laminin γ1 chain was selected as the best candidate among the polypeptides studied here for the incorporation into a collagen hydrogel. It was shown that the survival of NSCs was indeed promoted in the collagen hydrogel incorporating the heterodimer compared to the pure collagen hydrogel.

Variable disease severity in Saudi Arabian and Sudanese families with c.3924 + 2 T > C mutation of LAMA2

BackgroundCongenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene that encodes the laminin α2 chain, a component of the skeletal muscle extracellular matrix protein laminin-211. The clinical spectrum of the disease is more heterogeneous than previously thought, particularly in terms of motor achievement and disease progression. We investigated clinical findings and performed molecular genetic analysis in 3 families from Saudi Arabia and 1 from Sudan in whom congenital muscular dystrophy 1A was suspected based on homozygosity mapping and laminin α2 chain deficiency.MethodsWe investigated 9 affected individuals from 1 Sudanese and 3 Saudi families in whom MDC1A was suggested by clinical, neuroimaging and/or pathological findings and by homozygosity mapping at the LAMA2 locus. Morphological and immunohistochemical analysis were performed in 3 patients from the 3 Saudi families. SSCP analysis, DNA sequencing and microsatellite analysis were carried out in the 4 index cases.ResultsA previously described mutation in the LAMA2 gene, a homozygous T > C substitution at position +2 of the consensus donor splice site of exon 26, was found in the 4 index patients. Clinical evaluation of 9 patients from the 4 families revealed variable disease severity particularly as regards motor achievement and disease progression. Microsatellite analysis showed an identical mutation-associated haplotype in the 4 index cases indicating a founder effect of the mutation in all 4 families.ConclusionsOur data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical LAMA2-associated haplotype. The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.

Functional role of laminin α1 chain during cerebellum development

We had developed a conditional Laminin α 1 knockout-mouse model (Lama1cko) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1cko mice. Our study of the postnatal cerebellum of Lama1cko animals revealed a disrupted basement membrane correlated to an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.

Dilated cardiomyopathy with conduction defects in a patient with partial merosin deficiency due to mutations in the laminin‐α2‐chain gene: A chance association or a novel phenotype?

Patients with a partial reduction of merosin due to mutations in the laminin-α2 chain gene usually present with a mild form of congenital muscular dystrophy or a limb-girdle-like muscular dystrophy. To our knowledge, cardiac impairment has never been reported in such patients. A longitudinal study of a patient with partial laminin-α2 deficiency secondary to mutations in the LAMA2 gene revealed dilated cardiomyopathy with ventricular arrhythmias. Is this a chance association or a novel phenotype?

Autophagy is increased in laminin α2 chain-deficient muscle and its inhibition improves muscle morphology in a mouse model of MDC1A

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

Co-expression of laminin β3 and γ2 chains and epigenetic inactivation of laminin α3 chain in gastric cancer

Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that regulates cell adhesion and migration. Molecular alterations of LM-332 are involved in cancer progression. The aim of this study was to clarify alterations of LM-332 in gastric carcinoma. The expression of LM-332 subunits in 10 gastric carcinoma cell lines was investigated by RT-PCR, Western blotting, and immuno-cytochemical/immunofluorescent analyses. The promoter methylation status of LM-332-encoding genes (LAMA3, LAMB3 and LAMC2) was analyzed by methylation-specific PCR (MSP). The relationship between cell migration and LM-332 expression was assessed by the scratch assay. The expression of LM-332 was analyzed immunohistochemically in 90 gastric cancer tissues. Co-expression of laminin β3 and γ2 chains was often observed in gastric carcinoma cell lines at mRNA and protein levels. In contrast, there was no expression of laminin α3 at either the mRNA or protein levels. Extra-cellular secretion of laminin β3 and γ2 chains was found in 2 of the 10 cell lines. The LAMA3 gene was transcriptionally silenced by methylation of the promoter CpG islands in all of the cell lines, while the LAMB3 and LAMC2 genes were silenced in several cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored expression of the LM-332-encoding genes. Methylation frequency of LAMA3 was higher than those of the LAMB2 and LAMC2 genes in gastric cancer tissues. Migration distances were significantly correlated with cytoplasmic laminin γ2 chain expression. Immunohistochemistry showed frequent co-expression of laminin β3 and γ2 chains in gastric carcinoma cells, which was significantly correlated with depth of invasion and advanced tumor stage. The results suggest that the laminin β3 and γ2 chains accumulate intracellularly and play a role in gastric cancer progression, while epigenetic silencing of the laminin α3 chain may lead to inability to synthesize the basement membrane and may affect cancer cell invasion. Cancer cell motility appears to be associated with the cyto-plasmic laminin γ2 chain in vitro.

Effects of doxorubicin-encapsulating AG73 peptide-modified liposomes on tumor selectivity and cytotoxicity

Doxorubicin-encapsulating liposomal formulations, known as Doxil, have been used for the treatment of Kaposi’s sarcoma and ovarian cancer. However, there is still a need for a drug delivery system for doxorubicin that limits the treatment’s side effects, namely, mucositis and hand-and-foot syndrome. The AG73 peptide derived from the laminin α1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in some cancer cells. In this study, to develop a safer and more selective liposomal formulation, we prepared doxorubicin-encapsulating AG73 peptide-modified liposomes (AG73–Dox). First, we assessed the selectivity of AG73–Dox for cancer cells, including syndecan-2 over-expressing cells, using flow cytometry and confocal microscopy. AG73–Dox showed selective cellular uptake on cancer cells and enhancement of the intracellular uptake. Next, we examined the cytotoxicity of AG73–Dox using a WST assay. AG73–Dox exhibited a higher cytotoxicity against cancer cells than other control liposomes. In addition, we showed that the antitumor efficacy of AG73–Dox in vivo was better than that of free Dox. When we examined the biodistribution of liposomes, AG73 peptide-modified liposomes (AG73-L) tended to bind to intratumoral vessels and extravasated in the tumor tissue. Thus, further optimization of AG73-L toward tumor targeting may lead to a development of a useful tool for cancer therapy.

Abstract 431: Laminin-511 regulates melanoma cell function and tumor growth

Abstract Laminin family proteins are major non-collagenous components of basement membranes and have been found to be involved in modulating cell behavior. Laminin-511, composed of α5, β1, γ1 chains, is a major component of basement membrane of epithelia and blood vessels. Our lab has recently found increased expression of laminin-511 in malignant tumors. It has been found that tumor cells can synthesize and secrete laminin matrix to modify cellular environment and promote cell invasion. To explore the potential role of laminin-511 in melanoma, we blocked the expression of laminin-511 in human melanoma cell line SK-Mel-28 using RNA silencing strategy by transducing cells with lentiviral vector carrying laminin α5 chain specific shRNA (Lenti-Lα5 shRNA). Our data demonstrated that blocking the expression of laminin-511 significantly inhibited melanoma cell proliferation, attachment, migration and invasion in vitro, as well as tumor growth in vivo. In melanoma cells transduced with Lenti-Lα5 shRNA, compared with control melanoma cells, in cell proliferation analysis the total viable cells were 34.49% and 26.28% reduced on day 3 and day 5, respectively; in cell attachment assay the cell binding ability was decreased by 36.4%, 37.65% and 36.2% at 15, 30 and 60 minutes, respectively; in a chamber migration assay the cell migratory rate was dramatically down 90% at 24 hours; and in a Metrigel invasion assay the cell invasive capacity was markedly reduced by 89% at 24 hours. Furthermore, blocking laminin-511 expression significantly reduced tumor growth in vivo in nude mice. 18 tumors grew out of 20 injections with average volume 206 mm3 8 weeks post injection in cells tranduced with Lenti-Lα5 shRNA compared with 20 tumors out of 20 injections with average volume 590 mm3 in control cells. These findings revealed that laminin-511 plays important roles in melanoma cell function and tumor growth. In addition, our results strongly indicate laminin-511 a potential target for melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 431. doi:10.1158/1538-7445.AM2011-431

Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A

MDC1A, the second most prevalent form of congenital muscular dystrophy, results from laminin-α2 chain deficiency. This disease is characterized by extensive muscle wasting that results in extremely weak skeletal muscles. A large percentage of children with MDC1A are faced with respiratory as well as ambulatory difficulties. We investigated the effects of overexpressing insulin-like growth factor-1 (IGF-1) as a potential therapeutic target for the disease in the Lama2(Dy-w) mouse, a model that closely resembles human MDC1A. IGF-1 transgenic Lama2(Dy-w) mice showed increased survivability, body weight and muscle weight. In addition, these mice showed better ability to stand up on their hind limbs: a typical exploratory behavior seen in healthy mice. Histology and immunohistochemistry analyses revealed increased regenerative capacity and proliferation in IGF-1 transgenic Lama2(Dy-w) muscles. Western blot analysis showed increased phosphorylation of Akt and ERK1/2, both known to enhance myogenesis. Additionally, we saw increases in the expression of the regeneration markers MyoD, myogenin and embryonic myosin (myosin heavy chain 3, MYH3). We conclude that overexpression of IGF-1 in Lama2(Dy-w) mice increases lifespan and improves their overall wellbeing mainly through the restoration of impaired muscle regeneration, as fibrosis or inflammation was not impacted by IGF-1 in this disease model. Our results demonstrate that IGF-1 has a promising therapeutic potential in the treatment of MDC1A.

Transgenic Expression of Laminin α1 Chain Does Not Prevent Muscle Disease in the mdx Mouse Model for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder, and one of the most frequently encountered, but one for which there is as yet no treatment. Laminin-111 protein therapy was recently shown to be a promising approach to prevent muscle disease in the mdx mouse model of DMD. The present study demonstrated that transgenic expression of laminin α1 chain in mdx animals, resulting in laminin-111 heterotrimer formation in mdx muscle, does not improve the dystrophic phenotype. The mdx mice overexpressing laminin-111 (mdxLMα1) display features of mdx littermates: dystrophic pattern of muscle biopsy, elevated creatine kinase levels, reduced muscle strength, and decreased sarcolemmal integrity. Increased expression of integrin α7 is not beneficial for mdxLMα1 muscle, and components of the dystrophin-glycoprotein complex are not restored at the sarcolemma on laminin-111 overexpression. In summary, further studies are needed to verify the functionality of laminin-111 protein therapy in DMD and to describe the molecular events resulting from this approach.

A Possible Mechanism of Autoimmune‐Mediated infertility in Women with Endometriosis

Endometriosis has been proposed to be an autoimmune disease because of the presence of a variety of autoantibodies specific for endometrial or ovarian antigens. The object of the present study is to characterize binding specificity of anti-laminin-111 autoantibodies in infertile patients with endometriosis and to investigate whether these autoantibodies affect the in vitro embryo development. An ELISA analysis using overlapping synthesized peptides that covered the entire G domain of laminin-α1 chain was performed in infertile patients with endometriosis (n = 45). Mouse blastocysts were cultured in media containing the purified IgG from one antibody-positive serum on laminin-111-coated dishes. Anti-laminin-111 autoantibodies were directed to several particular biologically functional peptide sequences in laminin-α 1 chain G domain. The tested IgG significantly inhibited the extent of in vitro trophoblast outgrowth. Anti-laminin-111 autoantibodies may have major pathogenic roles on early reproductive failure including endometriosis-associated infertility.

Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies

Laminin-211 is a cell-adhesion molecule that is strongly expressed in the basement membrane of skeletal muscle. By binding to the cell surface receptors dystroglycan and integrin α7β1, laminin-211 is believed to protect the muscle fiber from damage under the constant stress of contractions, and to influence signal transmission events. The importance of laminin-211 in skeletal muscle is evident from merosin-deficient congenital muscular dystrophy type 1A (MDC1A), in which absence of the α2 chain of laminin-211 leads to skeletal muscle dysfunction. MDC1A is the commonest form of congenital muscular dystrophy in the European population. Severe hypotonia, progressive muscle weakness and wasting, joint contractures and consequent impeded motion characterize this incurable disorder, which causes great difficulty in daily life and often leads to premature death. Mice with laminin α2 chain deficiency have analogous phenotypes, and are reliable models for studies of disease mechanisms and potential therapeutic approaches. In this review, we introduce laminin-211 and describe its structure, expression pattern in developing and adult muscle and its receptor interactions. We will also discuss the molecular pathogenesis of MDC1A and advances toward the development of treatment.