Laminin Alpha2 Chain Research Articles

Single-Nucleus Landscape of Glial Cells and Neurons in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease with a projected significant increase in incidence. Therefore, this study analyzed single-nucleus AD data to provide a theoretical basis for the clinical development and treatment of AD. We downloaded AD-related monocyte data from the Gene Expression Omnibus database, annotated cells, compared cell abundance between groups, and investigated glial and neuronal cell biological processes and pathways through functional enrichment analysis. Furthermore, we constructed a global regulatory network for AD based on cell communication and ecological analyses. Our findings revealed increased abundance of Capping Protein Regulator And Myosin 1 linker 1 (CARMIL1)+ astrocytes (AST), Immunoglobulin Superfamily Member 21 (IGSF21)+ microglia (MIC), SRY-Box Transcription Factor 6 (SOX6)+ inhibitory neurons (InNeu), and laminin alpha-2 chain (LAMA2)+ oligodendrocytes (OLI) cell subgroups in tissues of patients with AD, while prostaglandin D2 synthase (PTGDS)+ AST, Src Family Tyrosine Kinase (FYN)+ MIC, and Proteolipid Protein 1 (PLP1)+ InNeu subgroups specifically decreased. We found that the cell phenotype of patients with AD shifted from a simpler to a more complex state compared to the control group. Cell communication analysis revealed strong communication between MIC and NEU. Furthermore, AST, MIC, NEU, and OLI were involved in oxidative stress- and inflammation-related pathways, potentially contributing to disease development. This study provides a theoretical basis for further exploring the specific mechanisms underlying AD.

Identification of established and novel extracellular matrix components in glioblastoma as targets for angiogenesis and prognosis.

Glioblastomas (GBM) are aggressive tumors known for their heterogeneity, rapid proliferation, treatment resistance, and extensive vasculature. Angiogenesis, the formation of new vessels, involves endothelial cell (EC) migration and proliferation. Various extracellular matrix (ECM) molecules regulate EC survival, migration, and proliferation. Culturing human brain EC (HBMEC) on GBM-derived ECM revealed a decrease in EC numbers compared to controls. Through in silico analysis, we explored ECM gene expression differences between GBM and brain normal glia cells and the impact of GBM microenvironment on EC ECM transcripts. ECM molecules such as collagen alpha chains (COL4A1, COL4A2, p < 0.0001); laminin alpha (LAMA4), beta (LAMB2), and gamma (LAMC1) chains (p < 0.0005); neurocan (NCAN), brevican (BCAN) and versican (VCAN) (p < 0.0005); hyaluronan synthase (HAS) 2 and metalloprotease (MMP) 2 (p < 0.005); MMP inhibitors (TIMP1-4, p < 0.0005), transforming growth factor beta-1 (TGFB1) and integrin alpha (ITGA3/5) (p < 0.05) and beta (ITGB1, p < 0.0005) chains showed increased expression in GBM. Additionally, GBM-influenced EC exhibited elevated expression of COL5A3, COL6A1, COL22A1 and COL27A1 (p < 0.01); LAMA1, LAMB1 (p < 0.001); fibulins (FBLN1/2, p < 0.01); MMP9, HAS1, ITGA3, TGFB1, and wingless-related integration site 9B (WNT9B) (p < 0.01) compared to normal EC. Some of these molecules: COL5A1/3, COL6A1, COL22/27A1, FBLN1/2, ITGA3/5, ITGB1 and LAMA1/B1 (p < 0.01); NCAN, HAS1, MMP2/9, TIMP1/2 and TGFB1 (p < 0.05) correlated with GBM patient survival. In conclusion, this study identified both established and novel ECM molecules regulating GBM angiogenesis, suggesting NCAN and COL27A1 are new potential prognostic biomarkers for GBM.

Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy.

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4+/+ and Lama4-/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4-/- recipients showed significantly increased platelet counts in blood compared to Lama4+/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4-/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1+ cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4-/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4-/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4-/- recipients (33.6%) than that in Lama4+/+ recipients (41.5%), raising a possibility that Lama4-/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4-/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

Interleukin-6 trans-signaling induced laminin switch contributes to reduced trans-endothelial migration of granulocytic cells

Background and aimsLaminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs). MethodsHUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively. ResultsStimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3. ConclusionsWe showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

Adipocyte-Specific Laminin Alpha 4 Deletion Preserves Adipose Tissue Health despite Increasing Adiposity.

Laminins are heterotrimeric glycoproteins with structural and functional roles in basement membranes. The predominant laminin alpha chain found in adipocyte basement membranes is laminin α4 (LAMA4). Global LAMA4 deletion in mice leads to reduced adiposity and increased energy expenditure, but also results in vascular defects that complicate the interpretation of metabolic data. Here, we describe the generation and initial phenotypic analysis of an adipocyte-specific LAMA4 knockout mouse (Lama4AKO). We first performed an in-silico analysis to determine the degree to which laminin α4 was expressed in human and murine adipocytes. Next, male Lama4AKO and control mice were fed chow or high-fat diets and glucose tolerance was assessed along with serum insulin and leptin levels. Adipocyte area was measured in both epididymal and inguinal white adipose tissue (eWAT and iWAT, respectively), and eWAT was used for RNA-sequencing. We found that laminin α4 was highly expressed in human and murine adipocytes. Further, chow-fed Lama4AKO mice are like control mice in terms of body weight, body composition, and glucose tolerance, although they have larger eWAT adipocytes and lower insulin levels. High-fat-fed Lama4AKO mice are fatter and more glucose tolerant when compared to control mice. Transcriptionally, the eWAT of high-fat fed Lama4AKO mice resembles that of chow-fed control mice. We conclude from these findings that adipocyte-specific LAMA4 deletion is protective in an obesogenic environment, even though overall adiposity is increased.

BiAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

Pseudomonas aeruginosa uses multiple receptors for adherence to laminin during infection of the respiratory tract and skin wounds

Pseudomonas aeruginosa efficiently adheres to human tissues, including the lungs and skin, causing infections that are difficult to treat. Laminin is a main component of the extracellular matrix, and in this study we defined bacterial laminin receptors on P. aeruginosa. Persistent clinical P. aeruginosa isolates from patients with cystic fibrosis, wounds or catheter-related urinary tract infections bound more laminin compared to blood isolates. Laminin receptors in the outer membrane were revealed by 2D-immunblotting, and the specificities of interactions were confirmed with ELISA and biolayer interferometry. Four new high-affinity laminin receptors were identified in the outer membrane; EstA, OprD, OprG and PA3923. Mutated bacteria devoid of these receptors adhered poorly to immobilized laminin. All bacterial receptors bound to the heparin-binding domains on LG4 and LG5 of the laminin alpha chain as assessed with truncated laminin fragments, transmission electron microscopy and inhibition by heparin. In conclusion, P. aeruginosa binds laminin via multiple surface receptors, and isolates from lungs of cystic fibrosis patients bound significantly more laminin compared to bacteria isolated from the skin and urine. Since laminin is abundant in both the lungs and skin, we suggest that laminin binding is an important mechanism in P. aeruginosa pathogenesis.

Molecular Links between Erythrocyte Adhesion and Vascular Dysfunction in Diabetes Mellitus, Polycythemia Vera, Retinal Vascular Occlusion

Despite clinical and epidemiological studies quantitative and qualitative abnormalities red blood cell (RBC) role in thrombosis has been ignored. Abnormal interaction of RBCs with vascular endothelium has been demonstrated in diabetes mellitus, Polycythemia vera (PV) and retinal vascular occlusion (RVO) after the first description in sickle cell anemia. The molecular basis has been identified. In diabetes mellitus, glycation of RBC band 3 caused the ligation to the receptor for advanced glycation end products (RAGE) present on endothelium. An abnormal Lu/BCAM (CD 239) expression in PV is responsible for the binding to endothelium annexin V. In RVO, RBC phosphatidylserine overexpression mediated the attachment to vascular laminin alpha-5 chain. Red blood cell binding altered the nonthrombotic properties of endothelium leading to thrombosis.

Abstract A07: Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model

Abstract Laminin-511 (composed of alpha5, beta1 and gamma1 chains) is a major extracellular matrix component in the basement membrane of human skin dermal microvascular endothelial cells (HDMECs). In this study the functions of laminin-511 in angiogenesis were investigated using in vitro knockdown and in vivo knockout skin model. In the in vitro studies, normal primary HDMECs and RNA interfering technique (siRNA) to silence the gene expression strategy were used. A lentiviral-based delivery system was developed that expresses laminin alpha5 chain specific siRNA (pL1) or control LUC siRNA (pLUC). HDMECs transduced with Laminin alpha5 siRNA (HDMEC/pL1) or control (HDMEC/pLUC) were analyzed in cell attachment, migration and a tubule formation assays. The mRNA expressions of laminin alpha5 and integrins were examined by real-time RT-PCR. Focal adhesion kinase (FAK) activation was studied by Western blot. In the in vivo study, a knockout mouse skin angiogenesis model was used. Since laminin alpha5 homozygous knockout (-/-) is embryonic lethal in mouse, skin from laminin alpha5 (-/-) embryos was transplanted onto the back of nude mouse, excisional wounds were created and wound angiogenesis was analyzed. Our results demonstrated that knockdown laminin alpha5 expression significantly inhibited the ability of endothelial cells in attachment, migration and tubule formation in vitro and blood vessel growth in vivo. Cell adhesive ability of HDMEC/pL1 was reduced 60% during a 60 minute attachment assay. Cell mobility of HDMEC/pL1 was found remarkably decreased 83% and 71% in a 12-hour and 48-hour chamber migration assay respectively; and in consistence, cell migration rates in a scratch migration assay were 56% and 53% reduced in HDMEC/pL1 cells at 48 and 72 hours respectively. Moreover, depletion of laminin alpha5 prevented HDMEC from forming enclosed capillary-like tubular structure at later stage in a three-dimensional tubule formation assay. While, in vivo, homozygous laminin alpha5 knockout skin was associated with significantly reduced growth of new blood vessels. A simultaneous and significant down regulation of integrins alphaV and beta3 was found correlated with the laminin alpha5 knockdown and reduced rate of cell migration. Further, cells with laminin alpha5 knockdown failed FAK activation, FAK phosphorylation on tyrosine 397 changed little in HDMEC/pL1 cells while there was markedly increase in control cells. In conclusion, our data suggested a possible involvement of laminin-511 in integrin alphaV and beta3-dependent angiogenesis and blood vessel maturation. Our works revealed the important roles of laminin-511 in endothelial cell activities, which proved its significance for angiogenesis. Citation Format: Jie Li, Tengjiao Cui. Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A07.

G.P.313: Intrafamilial heterogeneity in an alpha-dystroglycanopathy due to GDP-Mannose Pyrophosphorylase B (GMPPB) mutations

We describe two brothers who presented with different phenotypes and progression, sharing compound heterozygous mutations in the GMPPB gene. The index case, 34 years old, was referred to the muscle clinic for increased CK (>2.000), proximal weakness and calf hypertrophy. He is on therapy for myoclonic epilepsy following encephalitis in infancy and has mild cognitive and motor delay. Because of his symptoms, a muscle biopsy was performed: the immune-histochemistry showed a reduction in alpha dystroglycan labelling, with a secondary reduction of laminin alpha2 chain on western blot. Genes known to be associated to alpha-dystroglycanopathy have been excluded by direct sequencing (FKRP,POMT1, POMT2, POMGnT1,LARGE, FKTN). Two heterozygous variants in the GMPPB gene, a previously described mutation[c.860G>A (p.Arg287Gln)] and a novel variant [c.656T>C (p.Ile219Thr)]. The older brother, 36 years old, shares the same mutations having been shown to also have increased CK (2.832). He has global developmental delay presenting with speech and language difficulties, motor delay, impaired fine motor coordination and autistic spectrum disorder. He is not affected by weakness or fatigue, nor has weakness at physical examination. GMPPB catalyses the formation of GDP-mannose, required for O-mannosylation of alpha-dystroglycan. Mutations in this gene have been recently described in association to a spectrum of muscular dystrophy variants (Carss et al. AJHG, 2013), ranging from congenital to limb girdle muscular dystrophy, with additional symptoms as intellectual disability, epilepsy or brain structural anomalies. The family we describe highlights the clinical variability of these patients, with different presentation despite sharing the same mutations. Furthermore, it underlines the importance of testing CK in children presenting with global developmental delay: if the younger brother had not presented with a LGMD phenotype, the diagnosis in the older brother would have continued to be missed.

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

Molecular Basis for Dystroglycan Binding to Laminin‐G Domain‐Containing Ligands

Dystroglycan (DG) is a highly glycosylated extracellular matrix (ECM) receptor. The like‐acetylglucosaminyltransferase (LARGE) is responsible for posttranslational modification of alpha‐dystroglycan (α‐DG) which is required for its function. Reduced ligand binding activity of α‐DG due to perturbed glycosylation is a common pathologic feature among several different forms of muscular dystrophy (called dystroglycanopathies). We now report that LARGE is a bifunctional glycosyltransferase, with both xylosyltransferase (Xyl‐T) and glucuronyltransferase (GlcA‐T) activities. We examined the enzymatic activity of LARGE with respect to Xyl‐α‐pNP as well as GlcA‐β‐MU as the acceptors and UDP‐Xyl and UDP‐GlcA as donors, using a secreted form of LARGE that lacks its transmembrane domain (LARGEdTM). We have identified the enzymatic function of LARGE, which produces a polysaccharide with repeating units of [‐3Xyl‐α1,3GlcA‐β1‐] onto these acceptors. We further show that this modification confers binding ability on α‐DG to laminin‐G domain‐containing ECM ligands: laminin, agrin, and neurexin. α‐DG binds the LG4 and 5 domains of laminin alpha chains, and it is suggested that three basic patches contribute to these interactions. The LARGE‐synthesized, negatively charged glycan on α‐DG likely binds to laminin through electrostatic associations with these basic patches.

Laminin isoforms in atherosclerotic arteries from mice and man.

The properties of the arterial vasculature depend to a large extent on the activities of smooth muscle cells, which, in turn, are determined by their extracellular environment. During pathological conditions, such as atherosclerosis, this interaction is altered. In close proximity to medial smooth muscle cells are basement membrane components, such as different isoforms of laminin. These proteins can have great impact on cellular function via interaction with cell surface integrins. However, knowledge of laminins in smooth muscle cell basement membranes during normal and pathological conditions is scarce. Therefore, we have analyzed the presence of laminin isoforms in atherosclerotic lesions of apolipoprotein E (ApoE)-deficient mice. Our study revealed that the laminin chain isotype composition within atherosclerotic plaque tissue was different from the chain composition in the media. In addition, obvious differences in laminin chain composition could be observed in areas of the media, which were or were not associated with plaque tissue. Our major findings demonstrate that laminin gamma3 was exclusively present in media associated with plaque tissue. Laminin alpha2 was also enriched in these medial areas. Plaque tissue was predominantly enriched in laminin alpha5 chains. This general distribution applied to lesions both with and without a fibrous cap-like structure. The differential distribution of laminin chains were partially accompanied by changes in the presence of the integrin alpha subunits 7 and V. The distribution of laminin chains in human atherosclerotic arteries, with different size and morphology, grossly resembled their distribution in mouse arteries.

P13 Investigating pathophysiology and therapeutic strategies in a mouse model of spinal and bulbar muscular atrophy (SBMA)

to glycosylated epitopes on ADG. Loss of these glycans, specifically that identified by IIH6, is associated with a group of neuromuscular disorders collectively termed the dystroglycanopathies. Fukutin related protein (FKRP) is one of six determined/putative enzymes implicated in the glycosylation of ADG. We have previously generated a FKRP knock-down mouse which displays a hypoglycosylation of ADG and reduced laminin alpha 2 immunostaining at the muscle sarcolemma, the pial basement membrane and the inner limiting membrane (ILM) of the eye. Of particular interest is the absence of either IIH6 or laminin alpha 2 at the ILM in control or FKRP mice indicating that a novel ADG axis is involved at this basement membrane. In this paper we present evidence of an altered deposition of several laminin alpha chains in the eye and brain and suggest that multiple ligand interactions contribute to the pathogenesis of the dystroglycanopathies. Furthermore we have evidence indicating that DG interacts with laminin alpha 1 in the eye and the brain and that levels of laminin alpha 1 gene expression are up-regulated as a result of FKRP mediated hypoglycosylation of ADG.

P11 A reduction in the expression of Fukutin-related protein leads to the altered deposition of multiple laminin alpha chains in a mouse model for Muscle Eye Brain disease

P11 A reduction in the expression of Fukutin-related protein leads to the altered deposition of multiple laminin alpha chains in a mouse model for Muscle Eye Brain disease

Distinct Roles for Laminin Globular Domains in Laminin α1 Chain Mediated Rescue of Murine Laminin α2 Chain Deficiency

BackgroundLaminin α2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin α1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin α2 chain, α1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin α1 chain is an excellent tool to distinguish between the roles of dystroglycan and integrins in the neuromuscular system.Methodology/Principal FindingsHere, we provide insights into the functions of laminin α1LG domains and the division of their roles in MDC1A pathogenesis and rescue. Overexpression of laminin α1 chain that lacks the dystroglycan binding LG4-5 domains in α2 chain deficient mice resulted in prolonged lifespan and improved health. Importantly, diaphragm and heart muscles were corrected, whereas limb muscles were dystrophic, indicating that different muscles have different requirements for LG4-5 domains. Furthermore, the regenerative capacity of the skeletal muscle did not depend on laminin α1LG4-5. However, this domain was crucial for preventing apoptosis in limb muscles, essential for myelination in peripheral nerve and important for basement membrane assembly.Conclusions/SignificanceThese results show that laminin α1LG domains and consequently their receptors have disparate functions in the neuromuscular system. Understanding these interactions could contribute to design and optimization of future medical treatment for MDC1A patients.

Transgenic overexpression of laminin α1 chain in laminin α2 chain–deficient mice rescues the disease throughout the lifespan

Several approaches to treat laminin alpha2 chain-deficient congenital muscular dystrophy (MDC1A) in mouse models have been undertaken. Most have shown promising results in young animals. However, older animals have only been characterized to some extent. Herein we analyze the lifespan of laminin alpha2 chain-deficient mice with transgenic overexpression of laminin alpha1 chain. Further outcome measures included internalized myonuclei, heart fibrosis, grip strength, and serum creatine kinase activity. We show that laminin alpha2-chain-deficient animals that overexpress laminin alpha1 chain survive to up to 1.5-2 years of age. Furthermore, they displayed improved skeletal and heart muscle morphology, near-normal muscle strength, and normalized creatine kinase levels. Such an improvement of the dystrophic phenotype that persists to old age has not been previously demonstrated in mice. Our findings hold promise with regard to the efficient treatment of MDC1A patients in the future.

Index Of Suspicion In The Nursery

A male infant who has trisomy 21 is born large for gestational age at 36 weeks' gestation to a 40-year-old G4P2 mother. The mother experienced gestational diabetes and hypertension during the pregnancy. The hospital course in the neonatal intensive care unit (NICU) is uneventful. The infant consumes human milk supplemented with formula every 3 hours. Due to decreased intake, nutritional supplementation by nasogastric tube is initiated. He requires 25 to 30 minutes to feed by mouth with chin support and pacing. There is minimal emesis with feedings until 9 days after birth, when he begins vomiting with each feeding. The amount of emesis varies, consists of undigested milk, and is otherwise nonbilious and nonbloody. The infant can tolerate only small amounts of his feedings by either mouth or nasogastric tube before vomiting, but he continues to have two to three bowel movements per day and good urine output. On physical examination, the baby has physical features consistent with trisomy 21, including epicanthal folds, a flat nasal bridge, macroglossia, Brushfield spots, widened space between the first and second toes, single palmar creases, and generalized hypotonia. There is no notable abdominal distention or palpable abdominal masses. The remainder of the examination yields normal results. A radiologic study helps to reveal the diagnosis. A term male infant is admitted to the NICU after difficulty feeding and respiratory distress at 5 hours of age. The baby was born to a 35-year-old G3T2P0A0L2 woman. All maternal laboratory results were negative during prenatal screening, although prenatal ultrasonography during the third trimester showed oligohydramnios and intrauterine growth restriction. The initial evaluation after delivery revealed an asymmetric, small-for-gestational age infant whose birthweight was 2,215 g and Apgar scores were 9 and 9 at 1 and 5 minutes, respectively. On physical examination, the infant exhibited generalized hypotonia, poor suck …

A comparative analysis of oncofetal fibronectin and tenascin-C incorporation in tumour vessels using human recombinant SIP format antibodies

Tumour angioneogenesis is associated with the reexpression of oncofetal fibronectin (oncFn) and tenascin-C (oncTn-C) splice variants, which may serve as targets for antibody-based pharmacodelivery. Knowledge of the vascular distribution and organization in different tumours is of importance for the understanding of tumour vessel formation and might be crucial for therapy. Therefore, human SIP format antibodies against Fn ED-A, Fn ED-B and Tn-C A and C splice domains were used for immunofluorescence labelling in renal, lung, oral, colon, breast and urinary bladder carcinoma specimens and in a renal carcinoma xenograft. The spatial relation to stroma, vessels and vascular basement membrane (vBM) was analysed including CD31 and laminin alpha4 chain antibodies. Renal cell carcinomas and atypical carcinoid of the lung revealed vessel-restricted oncFn and/or oncTn-C depositions; all other entities showed a variable stroma positivity including vessels. The individual pattern of oncFn/oncTn-C incorporation in the vBM depended on tumour type, vessel size and intratumoural heterogeneity. There was a stratification of the vessel wall showing luminal oncFn and extraluminal oncTn-C depositions. As shown in the xenograft, perivascular oncTn-C is provided by carcinoma cells. In conclusion, tumours differ in the pattern of Fn or Tn-C isoform positivity in the vessel wall, potentially representing a tumour type specific endothelial cell-tumour cell-stromal cell interaction. Carcinoma cells themselves are involved in vascular Tn-C matrix organization. Up to antigen distribution, Fn and Tn-C domain antibodies may serve as vehicles for antiangiogenetic and antifibrotic agents; oncFn/oncTn-C based targeting should be adapted individually.