LAMC2 regulates the proliferation, invasion, and metastasis of gastric cancer via PI3K/Akt signaling pathway.

OBJECTIVES: Gastric cancer (GC) is a prevalent malignant tumor widely distributed globally, exhibiting elevated incidence and fatality rates. The gene LAMC2 encodes the laminin subunit gamma-2 chain and is found specifically in the basement membrane of epithelial cells. Its expression is aberrant in multiple types of malignant tumors. This research elucidated a link between LAMC2 and the clinical characteristics of GC and investigated the potential involvement of LAMC2 in GC proliferation and advancement. MATERIALS AND METHODS: LAMC2 expressions were detected in GC cell lines and normal gastric epithelial cell lines via qRT-PCR. Silencing and overexpression of the LAMC2 were conducted by lentiviral transfection. A xenograft mouse model was also developed for in vivo analysis. Cell functional assays were conducted to elucidate the involvement of LAMC2 in cell growth, migration, and penetration. Further, immunoblotting was conducted to investigate the impact of LAMC2 on the activation of signal pathways after lentiviral transfection. RESULTS: In the findings, LAMC2 expression was markedly upregulated in GC cell lines as opposed to normal gastric epithelial cells. In vitro analysis showed that sh-LAMC2 substantially inhibited GC cell growth, migration, and invasion, while oe-LAMC2 displayed a contrasting effect. Xenograft tumor models demonstrated that oe-LAMC2 accelerated tumor growth via high expression of Ki-67. Immunoblotting analysis revealed a substantial decrease in various signaling pathway proteins, PI3K, p-Akt, and Vimentin levels upon LAMC2 knockdown, followed by increased E-cadherin expression. Conversely, its overexpression exhibited contrasting effects. Besides, epithelial-mesenchymal transition (EMT) was accelerated by LAMC2. CONCLUSION: This study provides evidence indicating that LAMC2, by stimulating signaling pathways, facilitated EMT and stimulated the progression of GC cells in laboratory settings and mouse models. Research also explored that the abnormal LAMC2 expression acts as a biomarker for GC.

A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery.

Purpose: The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Methods: Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). Results: The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3 days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. Conclusions: These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. Translational Relevance: The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.

Extracellular matrix marker LAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous and aggressive disease characterized by a high risk of mortality and poor prognosis. It has been reported that Laminin γ2 (LAMC2) is highly expressed in a variety of tumors, and its high expression is correlated with cancer development and progression. However, the function and mechanism by which LAMC2 influences TNBC remain unclear. METHODS: Kaplan-Meier survival analysis and Immunohistochemical (IHC) staining were used to examine the expression level of LAMC2 in TNBC. Subsequently, cell viability assay, wound healing and transwell assay were performed to detect the function of LAMC2 in cell proliferation and migration. A xenograft mouse model was used to assess tumorigenic function of LAMC2 in vivo. Luciferase reporter assay and western blot were performed to unravel the underlying mechanism. RESULTS: In this study, we found that higher expression of LAMC2 significantly correlated with poor survival in the TNBC cohort. Functional characterization showed that LAMC2 promoted cell proliferation and migration capacity of TNBC cell lines via up-regulating CD44. Moreover, LAMC2 exerted oncogenic roles in TNBC through modulating the expression of epithelial-mesenchymal transition (EMT) markers. Luciferase reporter assay verified that LAMC2 targeted ZEB1 to promote its transcription. Interestingly, LAMC2 regulated cell migration in TNBC via STAT3 signaling pathway. CONCLUSION: LAMC2 targeted ZEB1 via activating CD44/STAT3 signaling pathway to promote TNBC proliferation and migration, suggesting that LAMC2 could be a potential therapeutic target in TNBC patients.

Neovascularization by DPSC-ECs in a Tube Model for Pulp Regeneration Study.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.

Leptin Promotes Vasculogenic Mimicry in Breast Cancer Cells by Regulating Aquaporin-1.

Leptin is an obesity-related hormone that plays an important role in breast cancer progression. Vasculogenic mimicry (VM) refers to the formation of vascular channels lined by tumor cells. This study aimed to investigate the relationship between leptin and VM in human breast cancer cells. VM was measured by a 3D culture assay. Signal transducers and activators of transcription 3 (STAT3) signaling, aquaporin-1 (AQP1), and the expression of VM-related proteins, including vascular endothelial cadherin (VE-cadherin), twist, matrix metalloproteinase-2 (MMP-2), and laminin subunit 5 gamma-2 (LAMC2), were examined by Western blot. AQP1 mRNA was analyzed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Leptin increased VM and upregulated phospho-STAT3, VE-cadherin, twist, MMP-2, and LAMC2. These effects were inhibited by the leptin receptor-blocking peptide, Ob-R BP, and the STAT3 inhibitor, AG490. A positive correlation between leptin and AQP1 mRNA was observed and was confirmed by RT-PCR. Leptin upregulated AQP1 expression, which was blocked by Ob-R BP and AG490. AQP1 overexpression increased VM and the expression of VM-related proteins. AQP1 silencing inhibited leptin-induced VM and the expression of VM-related proteins. Thus, these results showed that leptin facilitates VM in breast cancer cells via the Ob-R/STAT3 pathway and that AQP1 is a key mediator in leptin-induced VM.

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair.

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

Content and tissue expression of Collagen I, Collagen IV, and Laminin in the Extracellular Matrix in Prostate Adenocarcinoma.

Prostate cancer is one of the most common neoplasm in the male population. It is not known why some tumors become more aggressive than others. Although most studies show changes in the expression of cell adhesion molecules and the extracellular matrix correlated with the Gleason score, no study has objectively measured the tissue content of these molecules. This study aims to measure the content and tissue expression of collagen type I and IV and laminin in the extracellular matrix of patients with prostate adenocarcinoma and correlate these findings with the Gleason score and clinical characteristics. Forty-one patients who underwent radical prostate surgery at the Urology Department of a reference Hospital in Brazil between January 2015 and December 2020 were studied. The tissue protein content was estimated under light microscopy at a final magnification of 200 × . The mean collagen I score in prostate adenocarcinoma tissue samples was 7.16 ± 1.03 pixels/field. The mean type IV collagen score was 3.44 ± 0.61 pixels/field. The mean laminin score was 5.19 ± 0.79 pixels/field. The total Gleason score was correlated with both collagen and laminin. All the correlations were negative, which shows that the higher the collagen/laminin expression was, the lower the total Gleason score (p-value < 0,05). According to the Pearson correlation analysis, age has no statistical relationship with collagen and laminin content. PSA, in turn, showed a correlation only with laminin, but r = -0.378 (p = 0.015). Among the associated diseases and lifestyle habits, there is only statistical significance in the comparison of alcoholism for collagen I. For collagen IV and laminin, no statistical significance was obtained with the clinical variables analyzed.

Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model.

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

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.

Enhancing nerve regeneration in infraorbital nerve injury rat model: effects of vitamin B complex and photobiomodulation.

Orofacial nerve injuries may result in temporary or long-term loss of sensory function and decreased quality of life in patients. B vitamins are required for DNA synthesis and the repair and maintenance of phospholipids. In particular, vitamins B1, B6, and B12 are essential for neuronal function. Deficiency in vitamin B complex (VBC) has been linked to increased oxidative stress, inflammation and demyelination. Photobiomodulation (PBM) has antioxidant activity and is neuroprotective. In addition, a growing literature attests to the positive effects of PBM on nerve repair. To assess the effect of PBM and VBC on regenerative process we evaluated the expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), myelin basic protein (MBP), laminin and neurofilaments (NFs) using Western blotting to identify regenerative pattern after chronic constriction injury of the infraorbital nerve (CCI IoN) treated by PBM, VBC or its combination. After CCI IoN, the rats were divided into six groups naive, sham, injured (CCI IoN), treated with photobiomodulation (904 nm, 6.23 J/cm2, CCI IoN + PBM), treated with VBC (containing B1, B6 and B12) 5 times, CCI IoN + VBC) and treated with PBM and VBC (CCI IoN + VBC + PBM). The treatments could revert low expression of BDNF, MBP and laminin. Also reverted the higher expression of neurofilaments and enhanced expression of NGF. PBM and VBC could accelerate injured infraorbital nerve repair in rats through reducing the expression of neurofilaments, increasing the expression of BDNF, laminin and MBP and overexpressing NGF. These data support the notion that the use of PBM and VBC may help in the treatment of nerve injuries. This finding has potential clinical applications.

HIF-1α-mediated LAMC1 overexpression is an unfavorable predictor of prognosis for glioma patients: evidence from pan-cancer analysis and validation experiments.

BACKGROUND: Laminin subunit gamma-1 (LAMC1) is a major extracellular matrix molecule involved in the tumor microenvironment. Knowledge of the biological features and clinical relevance of LAMC1 in cancers remains limited. METHODS: We conducted comprehensive bioinformatics analysis of LAMC1 gene expression and clinical relevance in pan-cancer datasets of public databases and validated LAMC1 expression in glioma tissues and cell lines. The association and regulatory mechanism between hypoxia inducible factor-1α (HIF-1α) and LAMC1 expression were explored. RESULTS: LAMC1 expression in most cancers in The Cancer Genome Atlas (TCGA) including glioma was significantly higher than that in normal tissues, which had a poor prognosis and were related to various clinicopathological features. Data from the Chinese Glioma Genome Atlas also showed high expression of LAMC1 in glioma associated with poor prognoses. In clinical glioma tissues, LAMC1 protein was highly expressed and correlated to poor overall survival. LAMC1 knockdown in Hs683 glioma cells attenuated cell proliferation, migration, and invasion, while overexpression of LAMC1 in U251 cells leads to the opposite trend. Most TCGA solid cancers including glioma showed enhancement of HIF-1α expression. High HIF-1α expression leads to adverse prognosis in gliomas, besides, HIF-1α expression was positively related to LAMC1. Mechanistically, HIF-1α directly upregulated LAMC1 promotor activity. Hypoxia (2% O2)-treated Hs683 and U251 cells exhibited upregulated HIF-1α and LAMC1 expression, which was significantly attenuated by HIF-1α inhibitor YC-1 and accompanied by attenuated cell proliferation and invasion. CONCLUSIONS: High expression of LAMC1 in some solid tumors including gliomas suggests a poor prognosis. The hypoxic microenvironment in gliomas activates the HIF-1α/LAMC1 signaling, thereby promoting tumor progression. Targeted intervention on the HIF-1α/LAMC1 signaling attenuates cell growth and invasion, suggesting a new strategy for glioma treatment.

Decellularized Brain Extracellular Matrix Hydrogel Aids the Formation of Human Spinal-Cord Organoids Recapitulating the Complex Three-Dimensional Organization.

The intricate electrophysiological functions and anatomical structures of spinal cord tissue render the establishment of in vitro models for spinal cord-related diseases highly challenging. Currently, both in vivo and in vitro models for spinal cord-related diseases are still underdeveloped, complicating the exploration and development of effective therapeutic drugs or strategies. Organoids cultured from human induced pluripotent stem cells (hiPSCs) hold promise as suitable in vitro models for spinal cord-related diseases. However, the cultivation of spinal cord organoids predominantly relies on Matrigel, a matrix derived from murine sarcoma tissue. Tissue-specific extracellular matrices are key drivers of complex organ development, thus underscoring the urgent need to research safer and more physiologically relevant organoid culture materials. Herein, we have prepared a rat decellularized brain extracellular matrix hydrogel (DBECMH), which supports the formation of hiPSC-derived spinal cord organoids. Compared with Matrigel, organoids cultured in DBECMH exhibited higher expression levels of markers from multiple compartments of the natural spinal cord, facilitating the development and maturation of spinal cord organoid tissues. Our study suggests that DBECMH holds potential to replace Matrigel as the standard culture medium for human spinal cord organoids, thereby advancing the development of spinal cord organoid culture protocols and their application in in vitro modeling of spinal cord-related diseases.

Human Placenta Decellularized Extracellular Matrix Hydrogel Promotes the Generation of Human Spinal Cord Organoids with Dorsoventral Organization from Human Induced Pluripotent Stem Cells.

Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.

A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes.

BACKGROUND: The enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics. NEW METHOD: Development of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation. RESULTS: Myenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells. COMPARISON WITH EXISTING METHODS: This novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials. CONCLUSION: Our method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.

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.

A multi-ancestry GWAS of Fuchs corneal dystrophy highlights the contributions of laminins, collagen, and endothelial cell regulation.

Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation, but its molecular etiology remains poorly understood. We performed genome-wide association studies (GWAS) of FECD in the Million Veteran Program followed by multi-ancestry meta-analysis with the previous largest FECD GWAS, for a total of 3970 cases and 333,794 controls. We confirm the previous four loci, and identify eight novel loci: SSBP3, THSD7A, LAMB1, PIDD1, RORA, HS3ST3B1, LAMA5, and COL18A1. We further confirm the TCF4 locus in GWAS for admixed African and Hispanic/Latino ancestries and show an enrichment of European-ancestry haplotypes at TCF4 in FECD cases. Among the novel associations are low frequency missense variants in laminin genes LAMA5 and LAMB1 which, together with previously reported LAMC1, form laminin-511 (LM511). AlphaFold 2 protein modeling, validated through homology, suggests that mutations at LAMA5 and LAMB1 may destabilize LM511 by altering inter-domain interactions or extracellular matrix binding. Finally, phenome-wide association scans and colocalization analyses suggest that the TCF4 CTG18.1 trinucleotide repeat expansion leads to dysregulation of ion transport in the corneal endothelium and has pleiotropic effects on renal function.

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.

Specific nanoprobe design for MRI: Targeting laminin in the blood-brain barrier to follow alteration due to neuroinflammation.

Chronic neuroinflammation is characterized by increased blood-brain barrier (BBB) permeability, leading to molecular changes in the central nervous system that can be explored with biomarkers of active neuroinflammatory processes. Magnetic resonance imaging (MRI) has contributed to detecting lesions and permeability of the BBB. Ultra-small superparamagnetic particles of iron oxide (USPIO) are used as contrast agents to improve MRI observations. Therefore, we validate the interaction of peptide-88 with laminin, vectorized on USPIO, to explore BBB molecular alterations occurring during neuroinflammation as a potential tool for use in MRI. The specific labeling of NPS-P88 was verified in endothelial cells (hCMEC/D3) and astrocytes (T98G) under inflammation induced by interleukin 1ß (IL-1ß) for 3 and 24 hours. IL-1ß for 3 hours in hCMEC/D3 cells increased their co-localization with NPS-P88, compared with controls. At 24 hours, no significant differences were observed between groups. In T98G cells, NPS-P88 showed similar nonspecific labeling among treatments. These results indicate that NPS-P88 has a higher affinity towards brain endothelial cells than astrocytes under inflammation. This affinity decreases over time with reduced laminin expression. In vivo results suggest that following a 30-minute post-injection, there is an increased presence of NPS-P88 in the blood and brain, diminishing over time. Lastly, EAE animals displayed a significant accumulation of NPS-P88 in MRI, primarily in the cortex, attributed to inflammation and disruption of the BBB. Altogether, these results revealed NPS-P88 as a biomarker to evaluate changes in the BBB due to neuroinflammation by MRI in biological models targeting laminin.