Α5β1 Integrin Research Articles

Evaluation of VLA-4 (integrin α4β1) as a shared target for radiopharmaceutical therapy across solid tumors.

Radiopharmaceutical therapy (RPT) is a promising approach to treating solid tumors, but therapeutic advances are impeded by the lack of broadly expressed targets and shared molecular vulnerability across different tumor types. We evaluate VLA-4 (integrin α4β1) as a potential target for RPT in solid tumors using radiolabeled copper-64 ([64Cu]Cu-) and copper-67 ([67Cu]Cu-CB-TE1A1P-PEG4-LLP2A) LLP2A, a peptidomimetic ligand of VLA-4, for preclinical imaging and RPT testing. Expression analysis of ITGA4, encoding the alpha 4 subunit of VLA-4, revealed overexpression in hematological malignancies and various solid tumors compared to healthy tissue. Flow cytometry showed medium to high VLA-4 expression in 77% of tested cancer cell lines. PET/CT imaging with [64Cu]Cu-LLP2A demonstrated tracer uptake in tumors and lymphoid tissues. In toxicity studies, [67Cu]Cu-LLP2A administered at 37-74 MBq was well-tolerated, causing only thymic atrophy without long-term hematological or tissue toxicity. Tumor dose response was observed in B16-F10 melanoma models. Using orthotopic syngeneic models (B16-F10, B78, 4T1, GL261, TH-MYCN, and E2A-PBX1) and human cancer models (SK-MEL-37, 143B, and IMR-5), we conducted PET/CT imaging and biodistribution studies, with selected models used for [64Cu]Cu-LLP2A dosimetry calculations. Given VLA-4's broad expression across cancer tissues, demonstrated on-target effects, acceptable toxicity profile, and favorable dosimetry in preclinical models, further investigation of [67Cu]Cu-LLP2A as an RPT agent is warranted.

Immunohistochemical examination of PNAd, α4β1 integrin and MUC-2 expressions in the secretary phase endometrium of women diagnosed with recurrent implantation failure.

Successful embryo implantation is contingent upon the intricate interaction between the endometrium and the blastocyst. Recurrent implantation failure (RIF) signifies the clinical challenge of failing pregnancy post-transfer of high-quality embryos, fresh or frozen, in at least three in vitro fertilization (IVF) cycles, often in women under 40 years. Recent studies identify impaired blastocyst maternal tissue communication among recurrent implantation failure causes. Despite successful embryo transfer in vitro fertilization cycles, endometrial factors persist in women with recurrent implantation failure history, underscoring implantation's complexity. The implantation window, during which the endometrium becomes receptive to the blastocyst, involves the expression of key molecules that facilitate the implantation process. When the literature was examined, it was observed that comparative immunohistochemical studies on key molecules such as α4β1 integrin, MUC-2 and PNAd, which are thought to play a critical role in the endometrium before and during implantation, were limited. In this study, we aimed to investigate, through immunohistochemical and histological analyses, the roles of adhesion molecules in the secretory phase endometrium of patients diagnosed with RIF. Twenty-one patients diagnosed with recurrent implantation failure and 21 patients with a history of previous pregnancy at the Gynecology and Obstetrics Clinic of Balcalı Hospital, Faculty of Medicine, Çukurova University were clinically evaluated between days 19-21 of the menstrual cycle and included in the study. Endometrial biopsies, prepared for light and electron microscopy, received Hematoxylin and Eosin staining and anti-α4β1 integrin, anti-MUC-2, and anti-PNAd antibodies for immunohistochemistry. Blood samples were collected on the 2nd and 3rd days of the menstrual cycle to assess serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and thyroid-stimulating hormone (TSH). While FSH, LH, and E2 levels showed no significant difference, TSH was elevated in the recurrent implantation failure group. Structural differences in the endometrium included increased microvilli cells and reduced pinopod counts in infertile women compared to controls. In women with recurrent implantation failure MUC-2 expression were found to be elevated in the endometrial surface epithelium, while PNAd expression was reduced compared to the control group. These findings suggest structural and molecular disparities during the endometrial receptivity window in recurrent implantation failure women may underlie infertility by hindering blastocyst adherence. Molecular studies are needed to elucidate the pathophysiology of the biomarkers whose presence in the endometrium we examined immunohistochemically and to discover new molecules. Changing expressions of adhesion molecules (PNAd, α4β1 and MUC-2) under the influence of steroid hormones, remodeling of intercellular connections and stromal epithelial communication play an important role in endometrial receptivity.

Molecular and cellular regulators of embryo implantation and their application in improving the implantation potential of IVF-derived blastocysts.

In vitro fertilization (IVF) and embryo transfer (ET) are widely used in reproductive biology. Despite the transfer of high-quality blastocysts, the implantation rate of IVF-derived blastocysts remains low after ET. This article provides a comprehensive review of current research on embryo implantation regulators and their application to improve the implantation potential of IVF-derived blastocysts. The invivo mouse model revealed selective proteolysis immediately after expression in activated blastocysts, that is, degradation of ERα expression in activated blastocysts regulated by the ubiquitin-proteasome pathway, followed by completion of blastocyst implantation. Treatment of blastocysts to induce appropriate protein expression during invitro culture prior to ET is a useful approach for improving implantation rates. This approach showed that combined treatment with PRL, EGF, and 4-OH-E2 (PEC) improved the blastocyst implantation rates. Furthermore, arginine and leucine drive reactive oxygen species (ROS)-mediated integrin α5β1 expression and promote blastocyst implantation. Findings based on analysis of molecular and cellular regulators are useful for improving the implantation potential of IVF-derived blastocysts. These approaches may help to elucidate the mechanisms underlying the completion of the blastocyst implantation, although further investigation is required to improve the success of implantation and pregnancy.

Loss of Tyrosine Phosphatase Mu Promotes Scoliosis Progression Through Osteopontin-α5β1 Integrin Signaling and PIPK1γ90 Activity

Adolescent idiopathic scoliosis (AIS) is characterized by a curvature of the spine affecting approximately 4% of the pediatric population, and the mechanisms driving its progression remain poorly understood. Whole-exome sequencing of a French-Canadian AIS cohort with severe scoliosis identified rare variants in the PTPRM gene, which encodes Protein Tyrosine Phosphatase μ (PTPµ). However, these rare variants alone did not account for the pronounced reduction in PTPµ at both mRNA and protein levels in severe AIS cases. This led us to investigate epigenetic regulators and the identification of five microRNAs (miR-103a-3p, miR-107, miR-148a-3p, miR-148b-3p, and miR-152-3p) that target PTPRM mRNA. These microRNAs were significantly elevated in plasma from severe AIS patients, and miR-148b-3p was also upregulated in AIS osteoblasts. Phenotypic analysis of bipedal Ptrprm knockout (PTPµ −/−) mice showed increased prevalence and severity of scoliosis, while quadrupedal PTPµ −/− mice did not develop scoliosis, underscoring PTPµ’s role as a disease-modifying factor. Mechanistically, PTPµ deficiency was found to disrupt Gi-coupled receptor signaling in osteoblasts by enhancing the interaction between osteopontin (OPN) and α5β1 integrin, along with increased tyrosine phosphorylation of phosphatidylinositol-4-phosphate 5-kinase type I (PIPKIγ90). These findings provide novel insights into the molecular mechanisms underlying spinal deformity progression in AIS, linking PTPµ depletion to aberrant OPN-α5β1 integrin signaling and highlighting potential therapeutic targets to stop, mitigate, or prevent scoliosis.

P0528 Leukocyte Adhesive Function as Potential Predictive Marker for Treatment Response to Vedolizumab Among Bio-naive Patients with Crohn’s Disease

Abstract Background Vedolizumab (VDZ) is approved for the treatment of Crohn’s diseases (CD), but around 60% of patients fail to respond. Predicting response to VDZ, especially prior to treatment, is desirable but remains challenging. In this study, we investigated if α4β7 and α4β1 integrin activity may be used to predict response to VDZ. Methods Both α4β7 and α4β1 integrin activity were assessed using Leukocyte adhesive function assay (LAFA). LAFA uses microfluidic techniques to mimic blood circulation in vitro. Blood samples were fluorescently labelled and then pulled into microfluidic channels precoated with either MAdCAM-1 or VCAM-1 as substrates. Interactions between leukocytes and substrates was recorded and analysed by cell tracking software. Cell kinetic parameters were calculated and used for the assessment of α4β7 and α4β1 integrin activity. Mn2+ was applied as activator to assess the full potential of integrin activity. Bio-naive CD patients were recruited and all received VDZ as treatment. Treatment response was assessed at week 14 by endoscopy. Patients were classified as primary loss of response to VDZ if the simplified endoscopic score for CD at week 14 failed to decrease by 25% compared to baseline. Blood samples were collected at weeks 0 (prior to VDZ infusion), 2, 6 and 14 and then applied for LAFA. The differences in α4β7 and α4β1 integrin activity between VDZ responders and non-responders were prospectively assessed and a prediction model was subsequently established. Results A total of 27 patients were recruited and received VDZ. Twenty patients were classified as responders and seven as non-responders. No difference was found in α4β7 integrin activity on CD4 and CD8 cells between responders and non-responders, either before or after VDZ treatment. However, a lower α4β1 integrin activity was found on CD4 and CD8 cells among VDZ non-responders at week 0 compared with responders in the presence of Mn2+. Using the best LAFA-derived markers, a model to predict VDZ non-responders at week 0 was developed, in which a sensitivity of 0.90 and a specificity of 0.73 were achieved, a substantially better performance than the existing prediction tool CDST. In addition, α4β1 integrin activity on CD4 and CD8 cells among non-responders was gradually enhanced following subsequent VDZ treatment, which was not detected in responders (figure), implying a possible mechanism underlining primary loss of response to VDZ. Conclusion Patients with a low α4β1 integrin activity at week 0 which can be enhanced after VDZ, is likely to be primary non-responders to VDZ. Integrin activity measured by LAFA prior to treatment may be used as new diagnostic tool for the prediction VDZ response in bio-naïve CD patients. References Dulai PS, Boland BS, Singh S, et al. Development and Validation of a Scoring System to Predict Outcomes of Vedolizumab Treatment in Patients With Crohn’s Disease. Gastroenterology. 2018;155(3):687-695.e10. doi:10.1053/j.gastro.2018.05.039

P0815 Oral alpha 4 beta 7 (α4β7) integrin antagonist EA1080 (NSHO-101) demonstrates target engagement and α4β7 integrin receptor occupancy following once-daily administration in healthy volunteers

Abstract Background EA1080 (NSHO-101) is a selective, oral small molecule α4β7 integrin antagonist being advanced for the treatment of inflammatory bowel disease (IBD). EA1080 (NSHO-101) was safe and well tolerated and resulted in near complete inhibition of MAdCAM-1 binding to α4β7 integrin (receptor occupancy) on peripheral blood CD4+ T cells following twice-daily dosing with Formulation X in a healthy volunteer phase 1 study (DDW 2024: Su1789; NCT04223960). No effect on peripheral blood lymphocyte count was observed, confirming no specific engagement of α4β1 integrin. Here pharmacokinetics (PK) and pharmacodynamics (PD) results from a once-daily formulation are presented. Methods The PK and PD results from multiple ascending dose (MAD) cohorts with a formulation utilizing once-daily dosing were assessed in a healthy volunteer phase 1 study. Peripheral blood lymphocyte counts were assessed across the dosing period. Results Once-daily administration of EA1080 (NSHO-101) resulted in sustained, near-complete inhibition of MAdCAM-1 binding to α4β7 integrin on peripheral blood CD4+ T cells (≥95% receptor occupancy) across the dosing interval (24 hours). This correlated with plasma concentrations of active metabolite of EA1080 (EA1080-M) and approximated the level of receptor occupancy seen with twice-daily dosing with Formulation X. Plasma concentrations of EA1080-M reached steady-state levels within 3-4 days, and this correlated with maximal inhibition of MAdCAM-1 binding (receptor occupancy). No consistent trend in peripheral blood lymphocyte counts was observed across the dosing period. Conclusion These results demonstrate that once-daily oral administration of EA1080 (NSHO-101) results in near complete inhibition MAdCAM-1 binding to α4β7 integrin (≥95% receptor occupancy) across the dosing interval. No effect on peripheral blood lymphocyte counts was observed, confirming no specific engagement of α4β1. These results support continued development of a once-daily dosing formulation for future clinical studies.

P0010 Characterization of proliferating memory CD4⁺ T cells reveals a distinct immune signature in anti-α4β7 integrin therapy non-responders Inflammatory Bowel Disease patients

Abstract Background Failure to respond to the anti-α4β7 integrin therapy, vedolizumab, is observed in a subset of inflammatory bowel disease (IBD) patients, potentially linked to distinct T cell activation profiles. This study aims to characterize the transcriptional and phenotypic features of circulating memory CD4⁺ T cells in vedolizumab non-responders and their association with treatment resistance. Methods We performed multidimensional flow cytometry and single-cell RNA sequencing to analyze IBD patients' circulating memory CD4⁺ T cell compartment, comparing pre- and post-treatment peripheral blood samples from vedolizumab responders and non-responders. Proteogenomic analysis and TCR repertoire profiling of CD4⁺ T cells were conducted using matched peripheral blood samples. Flow cytometry characterized T cell subsets based on surface markers, migration receptors, transcription factors, and cytokine expression, focusing on differences in activation and migration profiles. Results Ki67⁺ cells were enriched in the memory CD4⁺ T cell population of vedolizumab non-responders in both UC and CD, confirmed by two independent cohorts. TCR repertoire analysis showed increased diversity in memory CD4⁺ T cells post-treatment. Single-cell sequencing and proteomics revealed these Ki67⁺ cells had a high activation and proliferation profile (HLA-DR, CD40LG, MKI67) with reduced IL7R and SELL expression. They displayed elevated integrins (α4, β1, β7) and gut-homing receptors (CXCR3, CCR6). Upregulated T-bet, EOMES, and RORγt and increased IL-17A, indicated a pro-inflammatory Th1/Th17 phenotype in non-responders. Conclusion Ki67⁺ memory CD4⁺ T cells are significantly enriched in the peripheral blood of vedolizumab non-responders, displaying a distinct transcriptional and phenotypic profile characterized by Th1/Th17 polarization, high integrin α4β1 expression, and enhanced gut-homing capacity. These findings indicate that proliferating effector memory CD4⁺ T cells could contribute to therapy resistance in IBD, emphasizing the need for alternative therapeutic strategies targeting these cells in anti-α4β7 integrin therapy non-responders.

Exploring the regulatory impact of insulin on type I collagen synthesis in hepatic stellate cells through α5β1 integrin

In the present study by Dodig and colleagues, published in Metabolism and Target Organ Damage , the authors investigate the role of insulin in promoting type I collagen synthesis in hepatic stellate cells (HSCs) through α5β1 integrin signaling. Using L-SACC1 transgenic mice, which exhibit hyperinsulinemia and insulin resistance without fasting hyperglycemia, the researchers demonstrate that elevated insulin levels significantly increase type I collagen production in HSCs. This effect is mediated by α5β1 integrin signaling rather than the PI3 kinase pathway. These findings suggest that chronic hyperinsulinemia may preprogram HSCs for enhanced fibrogenesis following liver injury, contributing to advanced fibrosis associated with metabolic disorders such as metabolic dysfunction-associated steatosis liver disease (MASLD) and type 2 diabetes. It further suggests that chronic hyperinsulinemia increases the risk of significant fibrosis burden in chronic liver disease. In this commentary, the strengths and limitations of this study are discussed, along with the potential impact of these findings on current treatment strategies for insulin resistance, endogenous hyperinsulinemia, and exogenous hyperinsulinemia in the development of MASLD and disease progression to fibrosis, cirrhosis, and hepatocellular carcinoma.

Exploring Integrin α5β1 as a Potential Therapeutic Target for Pulmonary Arterial Hypertension: Insights From Comprehensive Multicenter Preclinical Studies.

Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PAs) and progressive increase in pulmonary vascular resistance leading to right ventricular failure. Although several drugs are approved for the treatment of PAH, mortality rates remain high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. The expression of the RGD (arginylglycylaspartic acid)-binding integrin α5β1 was assessed in PAs, PA smooth muscle cells, and PA endothelial cells from patients with PAH and controls using NanoString, immunoblotting, and Mesoscale Discovery assays. RNA sequencing was conducted to identify gene networks regulated by α5β1 inhibition in PAH PA smooth muscle cells. The therapeutic efficacy of α5β1 inhibition was evaluated using a novel small molecule inhibitor and selective neutralizing antibodies in Sugen/hypoxia and monocrotaline rat models, with validation by an external contract research organization. Comparisons were made against standard-of-care therapies (ie, macitentan, tadalafil) and sotatercept and efficacy was assessed using echocardiographic, hemodynamic, and histological assessments. Ex vivo studies using human precision-cut lung slices were performed to further assess the effects of α5β1 inhibition on pulmonary vascular remodeling. We found that the arginine-glycine-aspartate RGD-binding integrin α5β1 is upregulated in PA endothelial cells and PA smooth muscle cells from patients with PAH and remodeled PAs from animal models. Blockade of the integrin α5β1 or depletion of the α5 subunit downregulated FOXM1 (forkhead box protein M1)-regulated gene networks, resulting in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. We demonstrated that α5β1 integrin blockade safely attenuates pulmonary vascular remodeling and improves hemodynamics and right ventricular function and matched or exceeded the efficacy of standard of care and sotatercept in multiple preclinical models. Ex vivo studies further validated its potential in reversing advanced remodeling in human precision-cut lung slices. These findings establish α5β1 integrin as a pivotal driver of PAH pathology and we propose its inhibition as a novel, safe, and effective therapeutic strategy for PAH.

Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis.

Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.

Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis

Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.

Structural and functional characterization of integrin α5-targeting antibodies for anti-angiogenic therapy.

Integrins are a large family of heterodimeric receptors important for cell adhesion and signaling. Integrin α5β1, also known as the fibronectin receptor, is a key mediator of angiogenesis and its dysregulation is associated with tumor proliferation, progression, and metastasis. Despite numerous efforts, α5β1-targeting therapeutics have been unsuccessful in large part due to efficacy and off-target effects. To mediate activation and signaling, integrins undergo drastic conformational changes. However, how therapeutics influence or are affected by integrin conformation remains incompletely characterized. Using cell biology, biophysics, and electron microscopy, we shed light on these relationships by characterizing two potentially therapeutic anti-α5β1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind α5β1 with nanomolar affinity and reduce angiogenesis in vitro . We demonstrate BIIG2 reduces tumor growth in two human xenograft mouse models and exhibits a strong specificity for connective tissue-resident fibroblasts and melanoma cells. Using electron microscopy, we map out the molecular interfaces mediating the integrin-antibody interactions and reveal that although both antibodies have overlapping epitopes and block fibronectin binding via steric hindrance, the effect on the conformational equilibrium is drastically different. While MINT1526A constricts α5β1's range of flexibility, BIIG2 binds without restricting the available conformational states. These mechanistic insights, coupled with the functional analysis, guide which aspects should be prioritized to avoid off-target effects or partial agonism in the design of future integrin-targeted therapeutics.

DNA Aptamer-Polymer Conjugates for Selective Targeting of Integrin α4β1+ T-Lineage Cancers.

Selective therapeutic targeting of T-cell malignancies is difficult due to the shared lineage between healthy and malignant T cells. Current front-line chemotherapy for these cancers is largely nonspecific, resulting in frequent cases of relapsed/refractory disease. The development of targeting approaches for effectively treating T-cell leukemia and lymphoma thus remains a critical goal for the oncology field. Here, we report the discovery of a DNA aptamer, named HR7A1, that displays low nanomolar affinity for the integrin α4β1 (VLA-4), a marker associated with chemoresistance and relapse in leukemia patients. After truncation of HR7A1 to a minimal binding motif, we demonstrate elevated binding of the aptamer to T-lineage cancer cells over healthy immune cells. Using cryo-EM and competition studies, we find that HR7A1 shares an overlapping binding site on α4β1 with fibronectin and VCAM-1, which has implications for sensitizing blood cancers to chemotherapy. We last characterize barriers to in vivo aptamer translation, including serum stability, temperature-sensitive binding, and short circulation half-life, and synthesize an aptamer-polymer conjugate that addresses these challenges. Future work will seek to validate in vivo targeting of α4β1+ tumors with the conjugate, establishing an aptamer-based biomaterial that can be readily adapted for targeted treatment of T-cell malignancies.

Podocyte YAP ablation decreases podocyte adhesion and exacerbates FSGS progression through α3β1 integrin.

Severe proteinuria in focal segmental glomerulosclerosis (FSGS) is closely associated with decreased adhesion, and subsequent loss, of podocytes. Yes-associated protein (YAP) is a key transcriptional coactivator that plays a significant role in maintaining cellular homeostasis. However, its role in podocyte adhesion and its specific mechanism in FSGS progression remain unclear. In this study, an adriamycin (ADR)-induced FSGS model was established using podocyte-specific Yap knockout (KO) mice and control mice. These mice were further treated with Pyrintegrin, an agonist of α3β1 integrin, or a vehicle. Additionally, an ADR-induced FSGS model was constructed using podocyte-specific Itga3 KO mice, which were subsequently treated with 1-oleoyl lysophosphatidic acid (LPA), a YAP activator, or a vehicle. Our findings demonstrated that YAP was positively correlated with podocyte adhesion. Podocyte-specific Yap KO mice exhibited reduced levels of α3β1 integrin and podocyte adhesion. Yap KO aggravated the ADR-induced reduction in α3β1 integrin and podocyte adhesion, resulting in significantly increased segmental or global glomerulosclerosis and proteinuria. Notably, treatment with a β1 integrin agonist partially ameliorated the decrease of podocyte adhesion and the worsening FSGS progression caused by Yap KO. Mechanistically, YAP was found to transcriptionally regulate α3- and β1 integrin via transcriptional enhanced associate domain 3 (TEAD3), with TEAD3 binding to the promoter region of Itga3. Furthermore, Itga3 KO or knockdown abolished the beneficial effects of YAP activation on podocyte adhesion and FSGS progression. In conclusion, our results demonstrate that YAP regulates podocyte adhesion and FSGS progression through its transcriptional regulation of α3β1 integrin via TEAD3. This suggests that the YAP-TEAD3-α3β1 integrin axis may serve as a promising therapeutic target for FSGS. © 2024 The Pathological Society of Great Britain and Ireland.

Matrix-mediated activation of murine fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) are key cells promoting cartilage damage and bone loss in rheumatoid arthritis (RA). They are activated to assume an invasive and migratory phenotype. While mechanisms of FLS activation are unknown, evidence suggests that pre-damaged extracellular matrix (ECM) of the cartilage can trigger FLS activation. Integrin α11β1 might be involved in the activation, as it is increased in RA patients and hTNFtg mice, an RA mouse model. We treated murine chondrocytes with TNFα to produce a damaged, RA-like matrix. Comparison to healthy chondrocyte matrix revealed decreased ECM proteins, e.g. collagens and proteoglycans, increased matrix-degrading proteins and elevated levels of inflammatory cytokines. FLS responded to the damaged chondrocyte matrix with a matrix-remodeling and pro-inflammatory phenotype characterized by a gene signature involved in matrix degradation and increased production of CLL11 and CCL19. Damaged chondrocyte matrix stimulated increased Itga11 expression in FLS, correlating with the increased α11β1 amounts in RA patients. FLS deficient in integrin α11β1 released lower amounts of inflammation-associated cytokines. Our results demonstrate differences in healthy and RA-like chondrocyte ECM and distinctly different responses of wt FLS to damaged versus healthy ECM.

Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets

Matrigel®/BME®, a basement membrane-like preparation, supports long-term growth of epithelial 3D organoids from adult stem cells [T. Sato et al., Nature 459, 262-265 (2009); T. Sato et al., Gastroenterology 141, 1762-1772 (2011)]. Here, we show that interaction between Matrigel's major component laminin-111 with epithelial α6β1-integrin is crucial for this process. The outer membrane protein Invasin of Yersinia is known to activate multiple integrin-β1 complexes, including integrin α6β1. A C-terminal integrin-binding fragment of Invasin, coated on culture plates, mediated gut epithelial cell adhesion. Addition of organoid growth factors allowed multipassage expansion in 2D. Polarization, junction formation, and generation of enterocytes, goblet cells, Paneth cells, and enteroendocrine cells were stable over time. Sustained expansion of other human, mouse, and even snake epithelia was accomplished under comparable conditions. The 2D "organoid sheet" format holds advantages over the 3D "in gel" format in terms of imaging, accessibility of basal and apical domains, and automation for high-throughput screening. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel®/BME®.

SPP1-ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin-Dependent PINK1/PARKIN Pathway Blockade.

Low back pain (LBP) caused by nucleus pulposus degeneration and calcification leads to great economic and social burden worldwide. Unexpectedly, no previous studies have demonstrated the association and the underlying mechanism between nucleus pulposus tissue degeneration and calcification formation. Secreted Phosphoprotein 1 (SPP1) exerts crucial functions in bone matrix mineralization and calcium deposition. Here, a novel function of SPP1 is reported, namely that it can aggravate nucleus pulposus cells (NPs) degeneration by negatively regulating extracellular matrix homeostasis. The degenerated NPs have a higher mineralization potential, which is achieved by SPP1. Mechanistically, SPP1 can accelerate the degeneration of nucleus pulposus cells by activating integrin α5β1 (ITGα5/β1), aggravating mitochondrial damage and inhibiting mitophagy. SPP1-ITGα5/β1 axis inhibits mitophagy by PINK1/PARKIN pathway blockade. In conclusion, SPP1 activates ITGα5/β1 to inhibit mitophagy, accelerates NPs degeneration, and induces calcification, thereby leading to intervertebral disc degeneration (IVDD) and calcification, identifying the potentially unknown mechanism and relationship between IVDD and calcification. Important insights are provided into the role of SPP1 in nucleus pulposus calcification in IVDD by inducing nucleus pulposus cell senescence through inhibition of mitophagy and may help develop potential new strategies for IVDD treatment.

The novel ECM protein SNED1 mediates cell adhesion via the RGD-binding integrins α5β1 and αvβ3.

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by the RGD integrins α5β1 and αvβ3. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis.

O-GlcNAcylation stabilized WTAP promotes GBM malignant progression in an N6-methyladenosine-dependent manner.

Interactions between mesenchymal glioblastoma stem cells (MES GSCs) and myeloid-derived macrophages (MDMs) shape the tumor-immunosuppressive microenvironment (TIME), promoting the progression of glioblastoma (GBM). N6-methyladenosine (m6A) plays important roles in the tumor progression. However, the mechanism of m6A in shaping the TIME of GBM remains elusive. Single-cell RNA sequencing and bulk RNA-seq datasets were employed to identify the critical role of WTAP in interactions between MES GBM and MDMs. The biological function of WTAP was confirmed both in vitro and in vivo. Mechanistically, mass spectrum, RNA immunoprecipitation (RIP), and co-immunoprecipitation assays were conducted. Here, we identified that m6A methyltransferase Wilms' Tumor 1-Associated Protein (WTAP), whose protein stability could be synergistically enhanced via OGT-mediated O-GlcNAcylation and USP7-mediated de-ubiquitination, promoted LOXL2 m6A modification to enhance its mRNA stabilization in an IGF2BP2-dependent manner, upregulating secretion of LOXL2 protein (sLOXL2). sLOXL2 then interacted with integrin α5β1 on GSCs to activate FAK-ERK signaling, inducing mesenchymal transition of GSCs in an autocrine manner. Meanwhile, sLOXL2 also activated the integrin α5β1-FAK-ERK axis in MDMs, which promoted M2-like MDM phenotypes in a paracrine pathway, thereby contributing to T cell exhaustion to induce GBM immune escape. In translational medicine, combinations of the OGT inhibitor by targeting WTAP expression and the LOXL2 antagonist by disrupting MES GSC and MDM interactions showed favorable outcomes to the anti-PD1 immunotherapy. WTAP plays critical roles in mesenchymal transition of GSCs and formation of TIME, highlighting the therapeutic potential of targeting WTAP and its downstream effectors to enhance the efficacy of immunotherapy.

Integrin α6 and integrin β4 in exosomes promote lung metastasis of colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The mechanisms underlying metastasis, which contributes to poor outcomes, remain elusive. We used the Cancer Genome Atlas dataset to compare mRNA expression patterns of integrin α6 (ITGA6) and integrin β4 (ITGB4) in patients with CRC. We measured ITGA6 and ITGB4 expression levels in highly metastatic (i.e., HCT116 and SW620) and lowly metastatic (i.e., SW480 and Caco2) CRC cell lines. Exosomes were isolated from cell culture media and characterized using western blotting and nanoparticle analyses. The role of exosomes in lung metastasis was investigated using xenograft experiments in mice models, which received CRC cell injection and were treated with exosomes. ITGA6 and ITGB4 were significantly overexpressed in CRC tissues, and ITGA6 was associated with the American Joint Committee on Cancer (AJCC) stage and outcome. ITGA6 and ITGB4, as well as exosomal ITGA6 and ITGB4, were significantly more highly expressed in HCT116 and SW620 cells than in SW480 and Caco2 cells. The proliferation and tubulogenesis of vascular endothelial cells were markedly decreased by disruption of ITGA6 and ITGB4 but were markedly increased by ectopic expression of ITGA6 and ITGB4. Exosomal ITGA6 and ITGB4 promoted CRC metastasis to the lung in vivo. Taken together, our findings suggested that exosomal ITGA6 and ITGB4 displayed organotropism to the lung and upregulated proliferation and tubulogenic capacities, which might help reduce lung metastasis from CRC. These findings provided new insights into the mechanisms of CRC metastasis and provided novel potential therapeutic targets.