LAMC1 attenuates neuronal apoptosis via FAK/PI3K/AKT signaling pathway after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The poor prognosis in patients with subarachnoid hemorrhage (SAH) is often attributed to neuronal apoptosis. Recent evidence suggests that Laminin subunit gamma 1 (LAMC1) is essential for cell survival and proliferation. However, the effects of LAMC1 on early brain injury after SAH and the underlying mechanisms are unknown. The current study aimed to reveal the anti-neuronal apoptotic effect and the potential mechanism of LAMC1 in the rat and in the in vitro SAH models. METHODS: The SAH model of Sprague-Dawley rats was established by endovascular perforation. Recombinant LAMC1 (rLAMC1) was administered intranasally 30 min after modeling. LAMC1 small interfering RNA (LAMC1 siRNA), focal adhesion kinase (FAK)-specific inhibitor Y15 and PI3K-specific inhibitor LY294002 were administered before SAH modeling to explore the neuroprotection mechanism of rLAMC1. HT22 cells were cultured and stimulated by oxyhemoglobin to establish an in vitro model of SAH. Subsequently, SAH grades, neurobehavioral tests, brain water content, blood-brain barrier permeability, western blotting, immunofluorescence, TUNEL, and Fluoro-Jade C staining were performed. RESULTS: The expression of endogenous LAMC1 was markedly decreased after SAH, both in vitro and in vivo. rLAMC1 significantly reduced the brain water content and blood-brain barrier permeability, improved short- and long-term neurobehavior, and decreased neuronal apoptosis. Furthermore, rLAMC1 treatment significantly increased the expression of p-FAK, p-PI3K, p-AKT, Bcl-XL, and Bcl-2 and decreased the expression of Bax and cleaved caspase -3. Conversely, knockdown of endogenous LAMC1 aggravated the neurological impairment, suppressed the expression of Bcl-XL and Bcl-2, and upregulated the expression of Bax and cleaved caspase-3. Additionally, the administration of Y15 and LY294002 abolished the protective roles of rLAMC1. In vitro, rLAMC1 significantly reduced neuronal apoptosis, and the protective effects were also abolished by Y15 and LY294002. CONCLUSION: Exogenous LAMC1 treatment improved neurological deficits after SAH in rats, and attenuated neuronal apoptosis in both in vitro and in vivo SAH models, at least partially through the FAK/PI3K/AKT pathway.

Cardiac arrhythmia in individuals with steroid sulfatase deficiency (X-linked ichthyosis): candidate anatomical and biochemical pathways.

Circulating steroids, including sex hormones, can affect cardiac development and function. In mammals, steroid sulfatase (STS) is the enzyme solely responsible for cleaving sulfate groups from various steroid molecules, thereby altering their activity and water solubility. Recent studies have indicated that Xp22.31 genetic deletions encompassing STS (associated with the rare dermatological condition X-linked ichthyosis), and common variants within the STS gene, are associated with a markedly elevated risk of cardiac arrhythmias, notably atrial fibrillation/flutter. Here, we consider emerging basic science and clinical findings which implicate structural heart abnormalities (notably septal defects) as a mediator of this heightened risk, and propose candidate cellular and biochemical mechanisms. Finally, we consider how the biological link between STS activity and heart structure/function might be investigated further and the clinical implications of work in this area.

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Oligodendrocyte-derived laminin-γ1 regulates the blood-brain barrier and CNS myelination in mice.

Although oligodendrocytes (OLs) synthesize laminin-γ1, the most widely used γ subunit, its functional significance in the CNS remains unknown. To answer this important question, we generated a conditional knockout mouse line with laminin-γ1 deficiency in OL lineage cells (γ1-OKO). γ1-OKO mice exhibit weakness/paralysis and die by post-natal day 33. Additionally, they develop blood-brain barrier (BBB) disruption in the cortex and striatum. Subsequent studies reveal decreased major facilitator superfamily domain containing 2a expression and increased endothelial caveolae vesicles, but unaltered tight junction protein expression and tight junction ultrastructure, indicating a transcellular, rather than a paracellular, mechanism of BBB breakdown. Furthermore, significantly reduced OL lineage cells, OL precursor cells (OPCs), proliferating OPCs, and mature OLs are observed in γ1-OKO brains in a region-specific manner. Consistent with this finding, various defects in myelination are detected in γ1-OKO brains at biochemical and ultrastructural levels. Overall, these results highlight important roles of OL-derived laminin-γ1 in BBB maintenance and OL biology (proliferation, differentiation, and myelination).

Laminin Alpha 2 Enhances the Protective Effect of Exosomes on Human iPSC-Derived Cardiomyocytes in an In Vitro Ischemia-Reoxygenation Model.

Ischemic heart disease, a leading cause of death worldwide, manifests clinically as myocardial infarction. Contemporary therapies using mesenchymal stromal cells (MSCs) and their derivative (exosomes, EXOs) were developed to decrease the progression of cell damage during ischemic injury. Laminin alpha 2 (LAMA2) is an important extracellular matrix protein of the heart. Here, we generated MSC-derived exosomes cultivated under LAMA2 coating to enhance human-induced pluripotent stem cell (hiPSC)-cardiomyocyte recognition of LAMA2-EXOs, thus, increasing cell protection during ischemia reoxygenation. We mapped the mRNA content of LAMA2 and gelatin-EXOs and identified 798 genes that were differentially expressed, including genes associated with cardiac muscle development and extracellular matrix organization. Cells were treated with LAMA2-EXOs 2 h before a 4 h ischemia period (1% O2, 5% CO2, glucose-free media). LAMA2-EXOs had a two-fold protective effect compared to non-treatment on plasma membrane integrity and the apoptosis activation pathway; after a 1.5 h recovery period (20% O2, 5% CO2, cardiomyocyte-enriched media), cardiomyocytes treated with LAMA2-EXOs showed faster recovery than did the control group. Although EXOs had a protective effect on endothelial cells, there was no LAMA2-enhanced protection on these cells. This is the first report of LAMA2-EXOs used to treat cardiomyocytes that underwent ischemia-reoxygenation injury. Overall, we showed that membrane-specific EXOs may help improve cardiomyocyte survival in treating ischemic cardiovascular disease.

Correlation of laminin subunit alpha 3 expression in pancreatic ductal adenocarcinoma with tumor liver metastasis and survival.

BACKGROUND: The high mortality rate of pancreatic ductal adenocarcinoma (PDAC) is primarily attributed to metastasis. Laminin subunit alpha 3 (LAMA3) is known to modulate tumor progression. However, the influence of LAMA3 on liver metastasis in PDAC remains unclear. This study aimed to elucidate whether LAMA3 expression is increased in PDAC with liver metastasis. PATIENTS AND METHODS: We extracted information related to LAMA3 expression levels and associated clinicopathological parameters from The Cancer Genome Atlas (TCGA) and four Gene Expression Omnibus (GEO) datasets. Clinicopathological analysis was performed; the Kaplan-Meier Plotter was used to evaluate LAMA3's prognostic effect in PDAC. We retrospectively collected clinicopathological data and tissue specimens from 117 surgically treated patients with PDAC at the Affiliated Hospital of Qingdao University. We assessed LAMA3 expression and investigated its correlation with the clinicopathological traits, clinical outcomes, and hepatic metastasis. RESULTS: Amplified expression of LAMA3 was observed in PDAC tissue compared with normal tissue in the TCGA and GEO databases. High LAMA3 expression was associated with poor overall survival (OS) and relapse-free survival (RFS) in patients with PDAC. LAMA3 expression was significantly enhanced in PDAC tissues than in adjacent tissues. Tumor tissues from patients with PDAC exhibiting liver metastasis showed higher LAMA3 expression than those without liver metastasis. High LAMA3 expression correlated with large tumor size and TNM stage. LAMA3 expression and liver metastasis were independent predictive factors for OS; the former was independently associated with liver metastasis. CONCLUSIONS: LAMA3 expression is elevated in patients with PDAC with liver metastasis and is a predictor of prognosis.

Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen -type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.

Nidogen in development and disease.

Nidogen, also known as entactin, is a multifunctional glycoprotein that plays a crucial role in the maintenance of the basement membrane (BM), morphogenesis and neuronal plasticity. This review aims to provide an overview of the structural features, molecular interactions and diverse functions associated with Nidogen. As a bridging molecule within the BM, Nidogen acts as a linchpin connecting various extracellular matrix (ECM) components. Its involvement in tissue development, homeostasis, and pathological conditions underscores its biological and medical significance. We discuss the current state of knowledge regarding Nidogen's role in tissue maintenance, cell adhesion, migration, and signaling, shedding light on its intricate contributions to physiological and pathological processes.

YAP/TAZ-mediated regulation of laminin 332 is enabled by ß4 integrin repression of ZEB1 to promote ferroptosis resistance.

We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin ß4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the ß4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the ß4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of ß4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of ß4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the ß4 integrin.

Characterization of glomerular basement membrane components within pediatric glomerular diseases.

Background: Disruptions in gene expression associated with the glomerular basement membrane (GBM) could precipitate glomerular dysfunction. Nevertheless, a comprehensive understanding of the characterization of GBM components within pediatric glomerular diseases and their potential association with glomerular function necessitates further systematic investigation. Methods: We conducted a systematic analysis focusing on the pathological transformations and molecular attributes of key constituents within the GBM, specifically Collagen IV α3α4α5, Laminin α5ß2γ1, and Integrin α3ß1, across prevalent pediatric glomerular diseases. Results: We observed upregulation of linear expression levels of COL4A3/4/5 and Laminin 5α proteins, along with a partial reduction in the linear structural expression of Podocin in idiopathic nephrotic syndrome (INS), encompassing minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), but showing a reduction in IgA nephropathy (IgAN), IgA vasculitis nephritis (IgAVN) and lupus nephritis (LN). Furthermore, our study revealed reductions in Laminin ß2γ1 and Integrin α3ß1 in both primary and secondary childhood glomerular diseases. Conclusion: In INS, notably MCD and FSGS, there is a notable increase in the linear expression levels of COL4A3/4/5 and Laminin 5α proteins. In contrast, in IgAN, IgAVN, and LN, there is a consistent reduction in the expression of these markers. Furthermore, the persistent reduction of Laminin ß2γ1 and Integrin α3ß1 in both primary and secondary childhood glomerular diseases suggests a shared characteristic of structural alterations within the GBM across these conditions.

Laminin 511-E8, an autoantigen in IgG4-related cholangitis, contributes to cholangiocyte protection.

Background & Aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease. Anti-laminin 511-E8 autoantibodies have been identified in its pancreatic manifestation. Laminin 511-E8 promotes endothelial barrier function, lymphocyte recruitment, and cholangiocyte differentiation. Here, we investigate anti-laminin 511-E8 autoantibody presence in IRC, and mechanisms via which laminin 511 may contribute to cholangiocyte protection. Methods: Anti-laminin 511-E8 serum autoantibody positivity was assessed by ELISA. RNA sequencing and RT-qPCR were performed on human H69 cholangiocytes treated with recombinant laminin 511-E8. H69 cholangiocytes were subjected to shRNA knockdown targeting genes encoding laminin 511 (LAMA5, LAMB1, LAMC1) or treated with recombinant laminin 511-E8. Cholangiocellular bile acid influx was quantified radiochemically using 22,23-3H-glycochenodeoxycholic acid (GCDC). GCDC-induced apoptosis was determined by Caspase-3/7 assays. Cholangiocellular barrier function was assessed by FITC-Dextran permeability assays. Immunofluorescent staining of laminin 511 and claudin 1 was performed on extrahepatic bile duct tissue of control and anti-laminin 511-E8 positive individuals with IRC. Results: Seven out of 52 individuals with IRC had autoantibodies against laminin 511-E8. Recombinant laminin 511-E8 led to differential expression of genes involved in secretion, barrier function, and inflammation. Knockdown of laminin 511 constituents increased toxic bile acid permeation and GCDC-induced apoptosis. Laminin 511-E8 treatment decreased toxic bile acid permeation and dose-dependently alleviated GCDC-induced apoptosis. LAMA5 and LAMC1 knockdown increased transepithelial permeability. Laminin 511-E8 treatment reduced transepithelial permeability and prevented T lymphocyte-induced barrier dysfunction. Laminin 511 and claudin 1 staining patterns appeared altered in anti-laminin 511-E8 positive individuals with IRC. Conclusions: Laminin 511-E8 is an autoantigen in subsets of individuals with IRC. Laminin 511 enhances cholangiocellular barrier function and protects cholangiocytes against T lymphocyte-induced barrier dysfunction, toxic bile acid permeation and bile acid-induced apoptosis. Impact and implications: A subset of patients with IgG4-related cholangitis (IRC) has autoantibodies against laminin 511-E8. In human cholangiocytes, laminin 511 protects against (T lymphocyte-induced) epithelial barrier dysfunction and hydrophobic bile acids. Laminin 511 and claudin 1 staining may be altered in extrahepatic bile ducts of patients with IRC who are anti-laminin 511-E8 positive. This makes it tempting to speculate that a decreased epithelial barrier function with attraction of immune cells and impaired bicarbonate secretion as a result of dysfunction of laminin 511 by autoantibody binding could potentially be a common systemic pathogenic mechanism in a subset of patients with IgG4-RD.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Leucine-Rich Repeat in Polycystin-1 Suppresses Cystogenesis in a Zebrafish (Danio rerio) Model of Autosomal-Dominant Polycystic Kidney Disease.

Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5ß1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1's role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

PEDF-derived peptide protects against Amyloid-ß toxicity in vitro and prevents retinal dysfunction in rats.

Amyloid-beta (Aß), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aß42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aß42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aß-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aß42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aß, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aß42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aß42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aß42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aß42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aß42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aß42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aß42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aß42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aß42 in the retina and suggest concepts on the molecular mechanism of Aß retinal pathogenicity.

Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues.

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Exploring a Solvent Dependent Strategy to Control Self-Assembling Behavior and Cellular Interaction in Laminin-Mimetic Short Peptide based Supramolecular Hydrogels.

Self-assembled hydrogels, fabricated through diverse non-covalent interactions, have been extensively studied in regenerative medicines. Inspired from bioactive functional motifs of ECM protein, short peptide sequences have shown remarkable abilities to replicate the intrinsic features of the natural extracellular milieu. In this direction, we have fabricated two short hydrophobic bioactive sequences derived from the laminin protein i. e., IKVAV and YIGSR. Based on the substantial hydrophobicity of these peptides, we selected a co-solvent approach as a suitable gelation technique that included different concentrations of DMSO as an organic phase along with an aqueous solution containing 0.1 % TFA. These hydrophobic laminin-based bioactive peptides with limited solubility in aqueous physiological environment showed significantly enhanced solubility with higher DMSO content in water. The enhanced solubility resulted in extensive intermolecular interactions that led to the formation of hydrogels with a higher-order entangled network along with improved mechanical properties. Interestingly, by simply modulating DMSO content, highly tunable gels were accessed in the same gelator domain that displayed differential physicochemical properties. Further, the cellular studies substantiated the potential of these laminin-derived hydrogels in enhancing cell-matrix interactions, thereby reinforcing their applications in tissue engineering.

Endothelial and mural laminin-α5 contributes to neurovascular integrity maintenance.

BACKGROUND: Laminin-α5, a major component of the basal lamina, is predominantly synthesized by endothelial and mural cells (pericytes and vascular smooth muscle cells) in the CNS. Loss of laminin-α5 in either population fails to induce any abnormalities due to functional redundancy. Thus, the functional significance of laminin-α5 in neurovascular integrity remains unknown. Here, we hypothesize that ablation of laminin-α5 in both endothelial and mural cells increases neurovascular permeability. METHODS: The compound knockout mice were generated by crossing laminin-α5 floxed mice with Tie2-Cre and PDGFRß-Cre, which target endothelial cells and mural cells, respectively. Neurovascular permeability in these mutants was determined with both exogenous and endogenous tracers. Endothelial paracellular and transcellular permeability was assessed by examining the expression of tight junction proteins and transcytosis-associated proteins. In addition, transmission electron microscopy (TEM) was used to visualize tight junction ultrastructure and endothelial caveolae vesicles. Defects in pericytes and astrocytes were investigated by examining pericyte coverage/contact and astrocyte polarity. RESULTS: Elevated neurovascular permeability was observed in the mutants. Subsequent studies found increased Caveolin-1 and decreased major facilitator superfamily domain-containing protein 2a (MFSD2A) expression, but unaltered Claudin-5 or zonula occludens-1 (ZO-1) expression. Consistent with these results, mutant mice exhibited increased endothelial caveolae vesicle number with intact tight junction structure under TEM. Additionally, pericyte coverage and contact were also decreased in the mutant mice, while astrocyte polarity was unaffected. CONCLUSIONS: These results strongly indicate that endothelial and mural cell-derived laminin-α5 actively maintains neurovascular integrity via the transcellular rather than paracellular mechanism.

T-Cell Receptor Sequences Identify Combined Coxsackievirus-Streptococci Infections as Triggers for Autoimmune Myocarditis and Coxsackievirus-Clostridia Infections for Type 1 Diabetes.

Recent research suggests that T-cell receptor (TCR) sequences expanded during human immunodeficiency virus and SARS-CoV-2 infections unexpectedly mimic these viruses. The hypothesis tested here is that TCR sequences expanded in patients with type 1 diabetes mellitus (T1DM) and autoimmune myocarditis (AM) mimic the infectious triggers of these diseases. Indeed, TCR sequences mimicking coxsackieviruses, which are implicated as triggers of both diseases, are statistically significantly increased in both T1DM and AM patients. However, TCRs mimicking Clostridia antigens are significantly expanded in T1DM, whereas TCRs mimicking Streptococcal antigens are expanded in AM. Notably, Clostridia antigens mimic T1DM autoantigens, such as insulin and glutamic acid decarboxylase, whereas Streptococcal antigens mimic cardiac autoantigens, such as myosin and laminins. Thus, T1DM may be triggered by combined infections of coxsackieviruses with Clostridia bacteria, while AM may be triggered by coxsackieviruses with Streptococci. These TCR results are consistent with both epidemiological and clinical data and recent experimental studies of cross-reactivities of coxsackievirus, Clostridial, and Streptococcal antibodies with T1DM and AM antigens. These data provide the basis for developing novel animal models of AM and T1DM and may provide a generalizable method for revealing the etiologies of other autoimmune diseases. Theories to explain these results are explored.