Laminin-α4 Is Upregulated in Both Human and Murine Models of Obesity.
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ABSTRACT:
SUBMITTER: Goddi A
PROVIDER: S-EPMC8355986 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fibroblastic reticular cells (FRCs) play important roles in tolerance by producing laminin α4 (Lama4) and altering lymph node (LN) structure and function. The present study revealed the specific roles of extracellular matrix Lama4 in regulating LN conduits using FRC-specific KO mouse strains. FRC-derived Lama4 maintained conduit fiber integrity, as its depletion altered conduit morphology and structure and reduced homeostatic conduit flow. Lama4 regulated the lymphotoxin β receptor (LTβR) pathway, which is critical for conduit and LN integrity. Depleting LTβR in FRCs further reduced conduits and impaired reticular fibers. Lama4 was indispensable for FRC generation and survival, as FRCs lacking Lama4 displayed reduced proliferation but upregulated senescence and apoptosis. During acute immunization, FRC Lama4 deficiency increased antigen flow through conduits. Importantly, adoptive transfer of WT FRCs to FRC Lama4-deficient mice rescued conduit structure, ameliorated Treg and chemokine distribution, and restored transplant allograft acceptance, which were all impaired by FRC Lama4 depletion. Single-cell RNA sequencing analysis of LN stromal cells indicated that the laminin and collagen signaling pathways linked crosstalk among FRC subsets and endothelial cells. This study demonstrated that FRC Lama4 is responsible for maintaining conduits by FRCs and can be harnessed to potentiate FRC-based immunomodulation.
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Project description:The mechanisms by which tumors develop resistance to angiogenesis inhibitors, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. We developed three human lung adenocarcinoma murine models of resistance to the VEGF inhibitor bevacizumab and, using species-specific profiling, separately investigated tumor cell and stromal molecules associated with resistance. Gene expression changes associated with acquired resistance occurred predominantly in stromal (mouse) and not tumor (human) cells. Components of the EGFR and FGFR2 pathways were significantly upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Dual targeting of VEGF and EGFR pathways with bevacizumab and erlotinib, or the VEGFR/EGFR inhibitor vandetanib, reduced pericyte coverage and increased progression-free survival. These findings demonstrate that alterations in tumor stromal pathways, including EGFR and FGFR2, are associated with, and may contribute to VEGF inhibitor resistance and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens. To identify changes in stromal and tumor gene expression associated with acquired resistance to anti-VEGF therapy, we performed RNA microarray analyses comparing H1975 control (vehicle-treated) and BV-resistant (bevacizumab-treated until progression) xenografts (n=3 biologic replicates per group of treatment) using Illumina mouse (WG-6 v2) and human (WG-6 v3)-specific expression arrays. Probes in these arrays have been designed to minimize cross-species reactivity and consistent with this essentially no cross reactivity was observed in experiments mixing human and mouse cell lines.Total RNA was extracted from snap-frozen tissues using the mirVanaTM miRNA Isolation Kit (Ambion, Austin, TX) according to manufacturer’s protocol. Biotin labeled cRNA samples for hybridization were prepared by using Illumina Total Prep RNA Amplification Kit (Ambion Inc., Austin, TX). One microgram of total RNA was used for the synthesis of cDNA and followed by an amplification and biotin labeling. Each of 1.5 μg of biotinylated cRNAs was hybridized to both mouseWG-6 v2 and human WG-6v3 Expression BeadChips (Illumina®, San Diego, CA) at the same time, for analysis of murine and human transcriptomes. Signals were developed by Amersham fluorolink streptavidin-Cy3 (GE Health care Bio-Sciences, Little Chalfont, UK). Gene expression data were collected by using Illumina bead Array Reader confocal scanner (BeadStation 500GXDW; Illumina Inc.). Data were analyzed using the BRB-ArrayTools Version 3.7.0 Beta platform developed by Dr. Richard Simon and the BRB-Array Tools development team. A log base 2 transformation was applied to the data set prior to data normalization. A median array was selected as the reference array for normalization and statistical significance was set using a p<0.01. Genes differentially expressed between groups were determined applying univariate t-test with estimation of the false discovery rate (FDR). Genes were determined using selection criteria of a p<0.005 and a fold-change ≥1.5.
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Project description:Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.
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Project description:Lymph node (LN) fibroblastic reticular cells (FRCs) define LN niches and regulate lymphocyte homeostasis through producing diverse extracellular matrix (ECM) components. We examined the role of ECM laminin α4 (Lama4) using FRC-Lama4 conditional KO Pdgfrb-Cre-/- × Lama4fl/fl mice. Single-cell RNA-sequencing (scRNA-Seq) data showed the promoter gene Pdgfrb was exclusively expressed in FRCs. Depleting FRC-Lama4 reduced Tregs and dendritic cells, decreased high endothelial venules, impaired the conduit system, and downregulated T cell survival factors in LNs. FRC-Lama4 depletion impaired the homing of lymphocytes to LNs in homeostasis and after allografting. Alloantigen-specific T cells proliferated, were activated to greater degrees in LNs lacking FRC-Lama4, and were more prone to differentiate into effector phenotypes relative to the Treg phenotype. In murine cardiac transplantation, tolerogenic immunosuppression was not effective in FRC-Lama4 recipients, which produced more alloantibodies than WT. After lung transplantation, FRC-Lama4-KO mice had more severe graft rejection with fewer Tregs in their LNs. Overall, FRC-Lama4 critically contributes to a tolerogenic LN niche by supporting T cell migration, constraining T cell activation and proliferation, and promoting Treg differentiation. Hence, it serves as a therapeutic target for immunoengineering.
| S-EPMC9246384 | biostudies-literature
Project description:BackgroundSymptomatic acute schistosomiasis mansoni is a systemic hypersensitivity reaction against the migrating schistosomula and mature eggs after a primary infection. The mechanisms involved in the pathogenesis of acute schistosomiasis are not fully elucidated. Osteopontin has been implicated in granulomatous reactions and in acute hepatic injury. Our aims were to evaluate if osteopontin plays a role in acute Schistosoma mansoni infection in both human and experimentally infected mice and if circulating OPN levels could be a novel biomarker of this infection.Methodology/principal findingsSerum/plasma osteopontin levels were measured by ELISA in patients with acute (n = 28), hepatointestinal (n = 26), hepatosplenic (n = 39) schistosomiasis and in uninfected controls (n = 21). Liver osteopontin was assessed by immunohistochemistry in needle biopsies of 5 patients. Sera and hepatic osteopontin were quantified in the murine model of schistosomiasis mansoni during acute (7 and 8 weeks post infection, n = 10) and chronic (30 weeks post infection, n = 8) phase. Circulating osteopontin levels are increased in patients with acute schistosomiasis (p = 0.0001). The highest levels of OPN were observed during the peak of clinical symptoms (7-11 weeks post infection), returning to baseline level once the granulomas were modulated (>12 weeks post infection). The plasma levels in acute schistosomiasis were even higher than in hepatosplenic patients. The murine model mirrored the human disease. Macrophages were the major source of OPN in human and murine acute schistosomiasis, while the ductular reaction maintains OPN production in hepatosplenic disease. Soluble egg antigens from S. mansoni induced OPN expression in primary human kupffer cells.Conclusions/significanceS. mansoni egg antigens induce the production of OPN by macrophages in the necrotic-exudative granulomas characteristic of acute schistosomiasis mansoni. Circulating OPN levels are upregulated in human and murine acute schistosomiasis and could be a non-invasive biomarker of this form of disease.
| S-EPMC5068698 | biostudies-literature
Project description:BackgroundSepsis, the life-threatening host response to infection, is a major cause of mortality. Obesity increases vulnerability to sepsis; however, some degree of obesity may be protective, called the "obesity paradox". This scoping review systematically maps the literature on outcomes associated with diet-induced obesity and sepsis-induced organ injury, focusing on non-transgenic murine models.MethodsA literature search of primary articles was conducted from database inception to June 2023. Eligible articles compared diet-induced obesity to non-obese mice in sepsis models involving live pathogens. Two reviewers screened articles and extracted data on obesogenic and sepsis models utilized, and organ injury outcomes, including physiological dysfunction, histological alterations, and biochemical changes.ResultsSeventeen studies met eligibility criteria; 82% used male C57BL/6 mice, and 88% used cecal ligation and puncture to induce sepsis. Most studies used 60% high-fat diets compared to 10-16% fat in controls. Seven (64%) studies reported increased mortality in obese septic mice, one (9%) observed a decrease, and three (37%) found no significant difference. The liver, lungs, and kidneys were the most studied organs. Alanine transaminase results were inconclusive. Myeloperoxidase levels were increased in the livers of two studies and inconclusive in the lungs of obese septic mice. Creatinine and neutrophil gelatinase-associated lipocalin were elevated in obese septic mice.ConclusionsThere is variability in the methodology and measured outcomes in murine models of diet-induced obesity and sepsis and a lack of studies in female mice. The absence of standardized models has produced conflicting findings on the impact of obesity on sepsis outcomes.
| S-EPMC10884395 | biostudies-literature
Project description:The mechanisms by which tumors develop resistance to angiogenesis inhibitors, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. We developed three human lung adenocarcinoma murine models of resistance to the VEGF inhibitor bevacizumab and, using species-specific profiling, separately investigated tumor cell and stromal molecules associated with resistance. Gene expression changes associated with acquired resistance occurred predominantly in stromal (mouse) and not tumor (human) cells. Components of the EGFR and FGFR2 pathways were significantly upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Dual targeting of VEGF and EGFR pathways with bevacizumab and erlotinib, or the VEGFR/EGFR inhibitor vandetanib, reduced pericyte coverage and increased progression-free survival. These findings demonstrate that alterations in tumor stromal pathways, including EGFR and FGFR2, are associated with, and may contribute to VEGF inhibitor resistance and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.
2011-04-22 | GSE26644 | GEO
Project description:The mechanisms by which tumors develop resistance to angiogenesis inhibitors, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. We developed three human lung adenocarcinoma murine models of resistance to the VEGF inhibitor bevacizumab and, using species-specific profiling, separately investigated tumor cell and stromal molecules associated with resistance. Gene expression changes associated with acquired resistance occurred predominantly in stromal (mouse) and not tumor (human) cells. Components of the EGFR and FGFR2 pathways were significantly upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Dual targeting of VEGF and EGFR pathways with bevacizumab and erlotinib, or the VEGFR/EGFR inhibitor vandetanib, reduced pericyte coverage and increased progression-free survival. These findings demonstrate that alterations in tumor stromal pathways, including EGFR and FGFR2, are associated with, and may contribute to VEGF inhibitor resistance and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens. To identify changes in stromal and tumor gene expression associated with acquired resistance to anti-VEGF therapy, we performed RNA microarray analyses comparing H1975 control (vehicle-treated) and BV-resistant (bevacizumab-treated until progression) xenografts (n=3 biologic replicates per group of treatment) using Illumina mouse (WG-6 v2) and human (WG-6 v3)-specific expression arrays. Probes in these arrays have been designed to minimize cross-species reactivity and consistent with this essentially no cross reactivity was observed in experiments mixing human and mouse cell lines.Total RNA was extracted from snap-frozen tissues using the mirVanaTM miRNA Isolation Kit (Ambion, Austin, TX) according to manufacturer’s protocol. Biotin labeled cRNA samples for hybridization were prepared by using Illumina Total Prep RNA Amplification Kit (Ambion Inc., Austin, TX). One microgram of total RNA was used for the synthesis of cDNA and followed by an amplification and biotin labeling. Each of 1.5 μg of biotinylated cRNAs was hybridized to both mouseWG-6 v2 and human WG-6v3 Expression BeadChips (Illumina®, San Diego, CA) at the same time, for analysis of murine and human transcriptomes. Signals were developed by Amersham fluorolink streptavidin-Cy3 (GE Health care Bio-Sciences, Little Chalfont, UK). Gene expression data were collected by using Illumina bead Array Reader confocal scanner (BeadStation 500GXDW; Illumina Inc.). Data were analyzed using the BRB-ArrayTools Version 3.7.0 Beta platform developed by Dr. Richard Simon and the BRB-Array Tools development team. A log base 2 transformation was applied to the data set prior to data normalization. A median array was selected as the reference array for normalization and statistical significance was set using a p<0.01. Genes differentially expressed between groups were determined applying univariate t-test with estimation of the false discovery rate (FDR). Genes were determined using selection criteria of a p<0.005 and a fold-change ≥1.5.
2011-04-22 | E-GEOD-26644 | biostudies-arrayexpress
Project description:BackgroundLaminin alpha2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin alpha1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin alpha2 chain, alpha1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin alpha1 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 alpha1LG domains and the division of their roles in MDC1A pathogenesis and rescue. Overexpression of laminin alpha1 chain that lacks the dystroglycan binding LG4-5 domains in alpha2 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 alpha1LG4-5. 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 alpha1LG 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.
| S-EPMC2906511 | biostudies-literature