Integrin Inhibitor Research Articles

Antibody response to SARS-CoV2 before and after the third vaccination in patients with Inflammatory Bowel Disease.

Introduction Patients with inflammatory bowel disease (IBD) receiving immunosuppressive therapy might have an increased risk of developing a severe course of SARS-CoV-2 infection. The aim of this study was to investigate the development of antibodies in immunosuppressed patients with IBD compared to a healthy control group and to determine the effect of immunmodulators on the level of anti-SARS-CoV-2 IgG antibody levels before and after a third vaccination against SARS-CoV-2. Methods This is a single-center study with a retrospective observational design. Seventy-one IBD patients matched by propensity score to 71 healthy employees (control group) were included. Blood was taken from both groups at predetermined times before and after the third booster vaccination. Results All patients with IBD (n=71, 100%) received immunomodulatory therapy. The mean antibody level before the third vaccination was 1352.88 U/ml (SD= 1011.489) in the IBD group and were not lower compared to the control group (p= 0.088). Gender, age, and disease duration had no significant impact on the development of antibody levels. Patients with TNF-alpha blockers had significantly lower antibody titers (p=0.011) compared to the control group. Patients with integrin inhibitor therapy had significantly higher antibody titers (p=0.003) than the controls. After the third vaccination, an increase in antibody titers was recorded in all patients in the IBD group. Conclusion We recorded an antibody titer in all patients with IBD that was not significantly lower compared to healthy controls despite immunomodulatory therapy. The booster vaccination led to an increase in antibody levels in all patients with IBD.

Biosensor for integrin inhibition of mammalian cell adhesion and migration using micropatterned cell culture substrate and retroreflective optical signaling

Integrins are a family of transmembrane receptors that play a crucial role in cell adhesion and migration. Integrins can uniquely transduce biochemical signals bidirectionally across the membrane and physically link the cell-cell and cell-extracellular matrix (ECM) with ligand bonds. The arginyl-glycyl-aspartic acid (RGD) peptide motif is present in the ECM as a minimal recognition sequence for integrins. To leverage this property in cell-based therapy, RGD variants, such as cyclic-type RGDfK (c(RGDfK)), which share a similar structure with RGD but exhibit a higher affinity for integrins, have been developed. However, because most evaluation methods for newly developed RGD variants focus on affinity strength, tools for cellular effects are required. In this study, we developed a new platform that integrates micropatterned three-dimensional cell culture substrates with a non-spectroscopic optical analysis system to quantitatively analyze the effects of RGD variants on cell adhesion and migration. The specially micropatterned substrate provides a cell adhesive and migration area to provide a restricted analysis area. Owing to the characteristics of retroreflective Janus particles (RJPs), a non-spectroscopic optical analysis system provides long-term stable optical verification properties and a simple optical setup. These techniques were integrated to quantitatively determine the integrin inhibitory effect of various concentrations of RGD variant. To demonstrate the efficacy of the developed cellular level RGD variant testing platform, the model cell line L929 fibroblast and model RGD variant c(RGDfK) were analyzed ranging from 0 to 10 μM. The results showed that the developed system could effectively and quantitatively analyze the effects of RGD variants on cells across various concentrations.

Bexotegrast Shows Dose-dependent Integrin αvβ6 Receptor Occupancy in Lungs of Participants with Idiopathic Pulmonary Fibrosis: A Phase 2, Open-Label Clinical Trial.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by dyspnea and loss of lung function. Transforming growth factor-beta (TGF-β) activation mediated by αv integrins is central to the pathogenesis of IPF. Bexotegrast (PLN 74809) is an oral, once-daily, dual-selective inhibitor of αvβ6 and αvβ1 integrins under investigation for the treatment of IPF. Positron emission tomography (PET) using an αvβ6-specific PET tracer could confirm target engagement of bexotegrast in the lungs of participants with IPF. Objectives: This Phase 2 study (NCT04072315) evaluated αvβ6 receptor occupancy in the lung, as assessed by changes from baseline in αvβ6 PET tracer uptake, following single dose administration of bexotegrast to participants with IPF. Methods: In this open-label, single-center, single-arm study, adults with IPF received up to 2 single doses of bexotegrast, ranging from 60 to 320 mg with or without background IPF therapy (pirfenidone or nintedanib). At baseline and approximately 4 hours after each orally administered bexotegrast dose, a 60-minute dynamic PET/CT scan was conducted following administration of an αvβ6-specific PET probe ([18F]FP-R01-MG-F2). αvβ6 receptor occupancy by bexotegrast was estimated from the changes in PET tracer uptake following bexotegrast. Pharmacokinetics, safety, and tolerability of bexotegrast were also assessed. Results: Eight participants completed the study. Total and unbound plasma bexotegrast concentrations increased in a dose-dependent manner, and regional PET volume of distribution (VT) values decreased in a dose- and concentration-dependent manner. The VT data fit a simple saturation model, producing an unbound bexotegrast EC50 estimate of 3.32 ng/mL. Estimated maximum receptor occupancy was 35%, 53%, 71%, 88%, and 92% following single 60, 80, 120, 240, and 320-mg doses of bexotegrast, respectively. No treatment-emergent adverse events related to bexotegrast were reported. Conclusions: Dose- and concentration-dependent αvβ6 receptor occupancy by bexotegrast was observed by PET imaging, supporting once-daily 160 to 320 mg dosing to evaluate efficacy in clinical trials of IPF. Trial registration number: NCT04072315 Primary source of funding: Pliant Therapeutics, Inc. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Implications of GPIIB-IIIA Integrin and Liver X Receptor in Platelet-Induced Compression of Ovarian Cancer Multi-Cellular Spheroids

Background: Platelets have been shown to promote ovarian cancer; however, the mechanism is poorly understood. Previously, we demonstrated that platelets reduce the size and increase the density of multi-cellular ovarian cancer spheroids in cell cultures. The objectives of this study were to determine if platelet inhibitors could counteract these effects, and to explore the mechanisms involved. Methods: FDA-approved platelet inhibitors were screened for their abilities to alter platelet effects on ovarian cancer spheroids. Mass spectrometry was used to identify proteins significantly altered in cancer cells upon exposure to platelets. The effects of platelets and/or liver x receptor agonists or antagonists on LXR activity were measured using ES-2 ovarian cancer cells transduced with an LXR-reporter vector. Results: Eptifibatide, a GPIIB-IIIA integrin inhibitor, and dipyridamole, an adenosine reuptake inhibitor, reduced and enhanced platelet effects on ovarian cancer spheroids, respectively. Proteomic studies identified the LXR/RXR and integrin pathways as mediators of platelet effects on ovarian cancer, and downstream effectors of eptifibatide. Conclusions: Integrin pathways and their downstream LXR/RXR effectors are implicated in how platelets alter ovarian cancer spheroid morphology. These results support studying eptifibatide and LXR/RXR agonists as candidate drugs for repurposing as therapeutic strategies to counteract platelet promotion of ovarian cancer.

Extracellular Vesicle Mobility in Collagen I Hydrogels Is Influenced by Matrix-Binding Integrins.

Extracellular vesicles (EVs) are a diverse population of membrane structures produced and released by cells into the extracellular space for the intercellular trafficking of cargo molecules. They are implicated in various biological processes, including angiogenesis, immunomodulation, and cancer cell signaling. While much research has focused on their biogenesis or their effects on recipient cells, less is understood about how EVs are capable of traversing diverse tissue environments and crossing biological barriers. Their interactions with extracellular matrix components are of particular interest, as such interactions govern diffusivity and mobility, providing a potential basis for organotropism. To start to untangle how EV-matrix interactions affect diffusivity, we use high speed epifluorescence microscopy, single particle tracking, and confocal reflectance microscopy to analyze particle mobility and localization in extracellular matrix-mimicking hydrogels composed of collagen I. EVs are compared with synthetic liposomes and extruded plasma membrane vesicles to better understand the importance of membrane composition on these interactions. By treating EVs with trypsin to digest surface proteins, we determine that proteins are primarily responsible for EV immobilization in collagen I hydrogels. We next use a synthetic peptide competitive inhibitor to narrow down the identity of the proteins involved to argynylglycylaspartic acid (RGD) motif-binding integrins, which interact with unincorporated or denatured nonfibrillar collagen. Moreover, the effect of integrin inhibition with RGD peptides has strong implications for the use of RGD-peptide-based drugs to treat certain cancers, as integrin inhibition appears to increase EV mobility, improving their ability to infiltrate tissue-like environments.

S3897 Integrin Inhibition and Collagen Dynamics: Unraveling a Unique Case of Collagenous Colitis in a Patient With Ulcerative Colitis

S3897 Integrin Inhibition and Collagen Dynamics: Unraveling a Unique Case of Collagenous Colitis in a Patient With Ulcerative Colitis

Discovery and Development of Highly Potent and Orally Bioavailable Nonpeptidic αvβ6 Integrin Inhibitors.

A series of 3-aryl((S)-3-fluoropyrrolidin-1-yl)butanoic acids were developed as potent orally bioavailable αvβ6 integrin inhibitors. Starting from a zwitterionic peptidomimetic series optimized for inhaled administration, the balancing of potency and passive permeability to achieve suitable oral agents through modification and exploration of aryl substituents and pKa of the central cyclic amine is described. (S)-4-((S)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoic acid was found to have highly desirable oral pharmacokinetic profiles in rat, dog, and minipig, with low to moderate clearance (26%, 7%, and 18% liver blood flow, respectively), moderate volumes of distribution (3.6, 1.4, and 0.9 L/kg, respectively), high to complete oral bioavailabilities, high αvβ6 integrin potency of pIC50 of 8.0, and high solubility in physiological media (>2 mg/mL). Equating to the estimated human dose range of 10-75 mg b.i.d. to achieve 90% αvβ6 target engagement at Cmin, it was selected for further investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis.

Cancer-associated fibroblasts confer ALK inhibitor resistance in EML4-ALK -driven lung cancer via concurrent integrin and MET signaling.

Cancer-associated fibroblasts (CAFs) are associated with tumor progression and modulate drug sensitivity of cancer cells. However, the underlying mechanisms are often incompletely understood and crosstalk between tumor cells and CAFs involves soluble secreted as well as adhesion proteins. Interrogating a panel of non-small cell lung cancer (NSCLC) cell lines driven by EML4-ALK fusions, we observed substantial CAF-mediated drug resistance to clinical ALK tyrosine kinase inhibitors (TKIs). Array-based cytokine profiling of fibroblast-derived conditioned- media identified HGF-MET signaling as a major contributor to CAF-mediated paracrine resistance that can be overcome by MET TKIs. However, 'Cell Type specific labeling using Amino acid Precursors' (CTAP)-based expression and phosphoproteomics in direct coculture also highlighted a critical role for the fibronectin-integrin pathway. Flow cytometry analysis confirmed activation of integrin β1 (ITGB1) in lung cancer cells by CAF coculture. Treatment with pharmacological inhibitors, cancer cell-specific silencing or CRISPR-Cas9-mediated knockout of ITGB1 overcame adhesion protein-mediated resistance. Concurrent targeting of MET and integrin signaling effectively abrogated CAF-mediated resistance of EML4-ALK -driven NSCLC cells to ALK TKIs in vitro . Consistently, combination of the ALK TKI alectinib with the MET TKI capmatinib and/or the integrin inhibitor cilengitide was significantly more efficacious than single agent treatment in suppressing tumor growth using an in vivo EML4-ALK -dependent allograft mouse model of NSCLC. In summary, these findings emphasize the complexity of resistance-associated crosstalk between CAFs and cancer cells, which can involve multiple concurrent signaling pathways, and illustrate how comprehensive elucidation of paracrine and juxtacrine resistance mechanisms can inform on more effective therapeutic approaches.

Fitness Screens Map State-Specific Glioblastoma Stem Cell Vulnerabilities.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSCs) that persist after therapy and seed treatment refractory recurrent tumors. GBM tumors display a high degree of intra- and inter-tumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury-response. Drug targets for each subtype are needed to effectively target GBM. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs, we designed the GBM5K targeted gRNA library and performed fitness screens in a total of 30 patient-derived GSC cultures. The focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and elucidated the functional dependency gradient existing between the developmental and injury-response states. Developmental-specific fitness genes were enriched for transcriptional regulators of neurodevelopment, whereas injury-response-specific fitness genes were highlighted by several genes implicated in integrin and focal adhesion signaling. These context-specific vulnerabilities conferred differential sensitivity to inhibitors of β1 integrin, FAK, MEK and OLIG2. Interestingly, the screens revealed that the subtype-specific signaling pathways drive differential cyclin D (CCND1 vs. CCND2) dependencies between subtypes. These data provide biological insight and mechanistic understanding of GBM heterogeneity and point to opportunities for precision targeting of defined GBM and GSC subtypes to tackle heterogeneity.

Hypoxia-driven heterogeneous expression of α5 integrin in glioblastoma stem cells is linked to HIF-2α

Despite numerous molecular targeted therapies tested in glioblastoma (GBM), no significant progress in patient survival has been achieved in the last 20 years in the overall population of GBM patients except with TTfield setup associated with the standard of care chemoradiotherapy. Therapy resistance is associated with target expression heterogeneity and plasticity between tumors and in tumor niches. We focused on α5 integrin implicated in aggressive GBM in preclinical and clinical samples. To address the characteristics of α5 integrin heterogeneity we started with patient data indicating that elevated levels of its mRNA are related to hypoxia pathways. We turned on glioma stem cells which are considered at the apex of tumor formation and recurrence but also as they localize in hypoxic niches. We demonstrated that α5 integrin expression is stem cell line dependent and is modulated positively by hypoxia in vitro. Importantly, heterogeneity of expression is conserved in in vivo stem cell-derived mice xenografts. In hypoxic niches, HIF-2α is preferentially implicated in α5 integrin expression which confers migratory capacity to GBM stem cells. Hence combining HIF-2α and α5 integrin inhibitors resulted in proliferation and migration impairment of α5 integrin expressing cells. Stabilization of HIF-2α is however not sufficient to control integrin α5 expression. Our results show that AHR (aryl hydrocarbon receptor) expression is inversely related to HIF-2α and α5 integrin expressions suggesting a functional competition between the two transcription factors. Collectively, data confirm the high heterogeneity of a GBM therapeutic target, its induction in hypoxic niches by HIF-2α and suggest a new way to attack molecularly defined GBM stem cells.

Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

We have developed a bladder cancer-on-a-chip model which supports the 3D growth of cells and can be used to assess and quantify bladder cancer cell invasiveness in a physiologically appropriate environment. Three bladder cancer cell lines (T24, J82, and RT4) were resuspended in 50% Matrigel® and grown within a multi-channel organ-on-a-chip system. The ability of live cells to invade across into an adjacent 50% Matrigel®-only channel was assessed over a 2-day period. Cell lines isolated from patients with high-grade bladder cancer (T24 and J82) invaded across into the Matrigel®-only channel at a much higher frequency compared to cells isolated from a patient with low-grade cancer (RT4) (p < 0.001). The T24 and J82 cells also invaded further distances into the Matrigel®-only channel compared to the RT4 cells (p < 0.001). The cell phenotype within the model was maintained as assessed by cell morphology and immunohistochemical analysis of E-cadherin. Treatment with ATN-161, an α5β1 integrin inhibitor and well-known migrastatic drug, caused a dose-dependent decrease in the invasiveness of the J82 cells (p < 0.01). The combined data demonstrate that our bladder cancer-on-a-chip model supports the retention of the bladder cancer cell phenotype and can be used to reproducibly assess and quantify the invasiveness of live bladder cancer cells.

Future directions for xenotransplantation in lungs.

Advancements in preclinical xenotransplant studies have opened doors for clinical heart and kidney xenotransplantation. This review assesses recent progress in lung xenotransplantation research and its potential clinical implications. The efficacy of the humanized von Willebrand factor in reducing platelet sequestration in ex-vivo and in-vivo lung xenotransplant models was showcased. Combining human tissue factor pathway inhibitor and CD47 expression with selectin and integrin inhibition delayed neutrophil and platelet sequestration. Enhanced expression of human complement regulatory proteins and thrombomodulin in genetically engineered pig lungs improved graft survival by reducing platelet activation and modulating coagulation disruptions. Knocking out the CMAH gene decreased antibody-mediated inflammation and coagulation activation, enhancing compatibility for human transplantation. Furthermore, CMAH gene knockout in pigs attenuated sialoadhesin-dependent binding of human erythrocytes to porcine macrophages, mitigating erythrocyte sequestration and anemia. Meanwhile, in-vivo experiments demonstrated extended survival of xenografts for up to 31 days with multiple genetic modifications and comprehensive treatment strategies. Experiments have uncovered vital insights for successful xenotransplantation, driving further research into immunosuppressive therapy and genetically modified pigs. This will ultimately pave the way for clinical trials designed to improve outcomes for patients with end-stage lung disease.

Evidence from recent clinical trials in fibrotic interstitial lung diseases.

Idiopathic pulmonary fibrosis (IPF) is the prototype of fibrosing interstitial lung diseases. It is mirrored by progressive pulmonary fibrosis (PPF), an umbrella term which characterizes disease behavior of various fibrotic interstitial lung diseases with irreversible progression, accounting for loss of lung function, exercise intolerance and respiratory failure leading to early mortality. Pirfenidone and nintedanib halve the decline in lung function but do not halt disease progression. Since the publication in 2014 of pivotal pirfenidone and nintedanib studies, a number of clinical trials were conducted, many of them did not reach their primary endpoints. In IPF, promising phase 2 trials were followed by large phase 3 trials that did not confirm a favorable efficacy to tolerability favorable profile, including those with ziritaxestat, an autotaxin-1 inhibitor, zinpentraxin-alpha (human recombinant pentraxin-2), and the monoclonal antibody pamrevlumab targeting connective tissue growth factor. Nevertheless, newer compounds that hold promise are currently being evaluated in phase 3 or phase 2b randomized controlled trials, including: nerandomilast, a preferential phosphodiesterase 4B inhibitor; admilparant, a lysophosphatidic acid receptor antagonist; inhaled treprostinil, a prostacyclin agonist; and bexotegrast, a dual-selective inhibitor of αvβ6 and αvβ1 integrins. Nerandomilast, admilparant, inhaled treprostinil, and inhaled AP01 (pirfenidone), are currently studied in patients with PPF. Despite recent frustrating negative results, there is a growing portfolio of candidate drugs developed in both IPF and PPF.

Rhodium-catalyzed homo-coupling reaction of aryl Grignard reagents and its application for the synthesis of an integrin inhibitor.

A novel Rh-catalyzed one-pot homo-coupling reaction of aryl Grignard reagents was achieved. The reaction with bromobenzenes having an electron-donating group or a halogen substituent gave the corresponding homo-coupling products in good yields, although the reaction using heterocyclic or aliphatic bromides scarcely proceeded. A Rh(III)-bis(aryl) complex, which might be formed from RhCl(PPh3)3 and the aryl Grignard reagents, plays an important role in giving the homo-coupling products in this reaction. Furthermore, we applied the reaction to the synthesis of a novel inhibitor for integrins which is critical for several diseases.

Integrin β3 Reprogramming Stemness in HER2-Positive Breast Cancer Cell Lines.

HER2-positive breast cancer, characterised by overexpressed HER2 levels, is associated with aggressive tumour behaviour and poor prognosis. Trastuzumab is a standard treatment; however, approximately 50% of patients develop resistance within one year. This study investigates the role of ITGβ3 in promoting stemness and resistance in HER2-positive breast cancer cell lines (HCC1954 and SKBR3). The findings demonstrate that chronic exposure to trastuzumab upregulates stem cell markers (SOX2, OCT4, KLF4, NANOG, SALL4, ALDH, BMI1, Nestin, Musashi 1, TIM3, CXCR4). Given the documented role of RGD-binding integrins in drug resistance and stemness, we specifically investigated their impact on resistant cells. Overexpression of ITGβ3 enhances the expression of these stem cell markers, while silencing ITGβ3 reduces their expression, suggesting a major role for ITGβ3 in maintaining stemness and resistance. Further analysis reveals that ITGβ3 activates the Notch signalling pathway, known for regulating stem cell maintenance. The combination of trastuzumab and cilengitide, an integrin inhibitor, significantly decreases the expression of stem cell markers in resistant cells, indicating a potential therapeutic strategy to overcome resistance. These results identify the importance of ITGβ3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy.

A simplified G-CSF–free procedure allows for in vivo HSC gene therapy of sickle cell disease in a mouse model

AbstractWe have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4β1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice. Mobilization with WU-106 + AMD3100 in SCD mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of the prime editing vector together with in vivo selection resulted in ∼23% correction of the SCD mutation in the bone marrow and peripheral blood cells of SCD mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing frequencies were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.

Inhibition of alphaV integrins with cilengitide downregulates in vitro pro-fibrotic events in aortic smooth muscle cells and limits in vivo thoracic aortic aneurysm progression

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health "Ricerca Finalizzata - Giovani Ricercatori" Background The ‘greatest artery’, i.e., the aorta, can be affected by either acute conditions or chronic disorders, such as the aneurysm, which is the segmental aortic dilation with at least 50% increase in the normal diameter. Aortic dilation can often lead to lethal events, such as aortic dissection and/or vessel rupture. The aneurysms occurring in the thoracic trait of the aorta (TAA) may depend on comorbidities, such as hypertension (i.e., sporadic non-syndromic TAA, sTAA), or on genetically determined conditions, such as Marfan syndrome (MFS). To date, the surgical replacement of aorta represents the most effective therapeutic approach in all TAA patients, since an etiologic pharmacological treatment is still lacking. Thus, the identification of novel disease targets is mandatory. Integrin receptors are key players in cellular mechanotransduction responses. Specifically, integrins containing αV subunit have been described as important mechanotransduction mediators and responsible for pro-fibrotic molecular events. Recently, it has been reported the effectiveness of a broad αV integrin inhibitor in limiting the TAA progression by acting on FAK/AKT/mTORC signaling pathway in a MFS in vivo model. However, the specific αV integrin subgroup directly involved in this detrimental process is yet not known. Purpose We evaluated the effectiveness of a specific αVβ3 and αVβ5 integrin inhibitor (i.e., cilengitide) in downregulating pro-fibrotic in vitro events on vascular smooth muscle cells (VSMC) isolated from TAA patients’ aortic wall, and in limiting in vivo thoracic aortic dilation on a MFS mice model. Results By comparison with healthy control (HC)-VSMC, both sTAA-VSMC and MFS-VSMC showed higher levels of αVβ3 and αVβ5 integrins as well as pro-fibrotic collagen I. The in vitro inhibition of these two αV integrins mediated by cilengitide resulted more effective in collagen I downregulation when compared with GLPG0187 (pan αV inhibitor). Noteworthy, the cilengitide-mediated αV integrin inhibition consistently tempered pro-fibrotic phenotype of sTAA- and MFS-VSMC by directly acting on both canonical (p-SMAD2/3) and non-canonical (p-ERK1/2) TGF-β downstream pathways and by downregulating mechanotransduction mediators (e.g., RhoA GTPase, ROCK1, talin-1). The 2D-echocardiographic results on MFS mice treated with cilengitide confirmed the greater effectiveness of this drug in limiting the TAA progression, in comparison not only with vehicle but also with the broad integrin inhibitor GLPG0187. Conclusion Altogether, our results improve the know-how on αV integrin inhibition, highlighting the role of cilengitide as potential target for future therapeutic approaches to limit TAA progression, both in sporadic and in MFS patients.

The Role of αvβ3 Integrin in Cancer Therapy Resistance.

A relevant challenge for the treatment of patients with neoplasia is the development of resistance to chemo-, immune-, and radiotherapies. Although the causes of therapy resistance are poorly understood, evidence suggests it relies on compensatory mechanisms that cells develop to replace specific intracellular signaling that should be inactive after pharmacological inhibition. One such mechanism involves integrins, membrane receptors that connect cells to the extracellular matrix and have a crucial role in cell migration. The blockage of one specific type of integrin is frequently compensated by the overexpression of another integrin dimer, generally supporting cell adhesion and migration. In particular, integrin αvβ3 is a key receptor involved in tumor resistance to treatments with tyrosine kinase inhibitors, immune checkpoint inhibitors, and radiotherapy; however, the specific inhibition of the αvβ3 integrin is not enough to avoid tumor relapse. Here, we review the role of integrin αvβ3 in tumor resistance to therapy and the mechanisms that have been proposed thus far. Despite our focus on the αvβ3 integrin, it is important to note that other integrins have also been implicated in drug resistance and that the collaborative action between these receptors should not be neglected.

INHIBITION OF INTEGRIN VLA-3 AND TETRASPANIN CD151 PROTECTS AGAINST NEUTROPHIL-MEDIATED ENDOTHELIAL DAMAGE.

Background: The recruitment of neutrophils to sites of localized injury or infection is initiated by changes on the surface of endothelial cells located in proximity to tissue damage. Inflammatory mediators, such as TNF-α, increase surface expression of adhesive ligands and receptors on the endothelial surface to which neutrophils tether and adhere. Neutrophils then transit through the activated endothelium to reach sites of tissue injury with little lasting vascular injury. However, in cases of sepsis, the interaction of endothelial cells with highly activated neutrophils can cause damage vascular damage. The identification of molecules that are essential for neutrophil diapedesis may reveal targets of therapeutic opportunity for preservation of endothelial function in the presence of critical illness. We tested the hypothesis that inhibition of neutrophil β1 integrin very late antigen-3 (VLA-3; α3β1) and/or inhibition of the tetraspanin (TM4) family member CD151 would protect against neutrophil-mediated loss of endothelial function. Methods: Blood was obtained from septic patients or healthy donors. Neutrophils were purified, and aliquots were treated with/without proinflammatory molecules. Confluent human umbilical vascular endothelial cells were activated with TNF-α. Electric cell impedance sensing was used to determine monolayer resistance over time after the addition of neutrophils that were treated with blocking antibodies against VLA-3 and/or CD151 or isotype controls. Groups (depending on relevancy) were analyzed by Mann-Whitney U test, Wilcoxon test, or repeated-measures one-way ANOVA. Results: Neutrophils from septic patients and neutrophils activated ex vivo reduced endothelial monolayer resistance to a greater extent than neutrophils from healthy donors. Antibody blockade of VLA-3 and/or CD151 significantly reduced activation-associated endothelial damage. Similar findings were demonstrated on fibronectin, collagen I, collagen IV, and laminin, suggesting that neutrophil surface VLA-3 and CD151 are responsible for endothelial damage regardless of substrata and are likely to be operative in all bodily tissues. Conclusion: This report identifies VLA-3 and CD151 on the activated human neutrophil, which are responsible for damage to endothelial function. Targeting these molecules in vivo may demonstrate preservation of organ function during critical illness.

#2376 Tubular exosomes in renal ECM microenvironment promotes fibrosis progression by activating latent TGF-β1

Abstract Background and Aims Chronic kidney disease (CKD) is characterized with progressive fibrosis caused by excessive extracellular matrix (ECM) deposition. Tubular epithelial cells secreted increasing exosomes into extracellular space under pathological conditions with integrin as the prominent constituent membrane cargo. Due to the critical role of integrin in activation of TGF-β1 signalling, this study aims to investigate the performance of tubular-derived exosomes in the ECM microenvironment, exploring the potential mechanism of exosomal integrin mediated activation of latent TGF-β1. Method A fibrosis mouse model was established with unilateral ureteral obstruction (UUO) and an ITGβ6 overexpression cell line were established to observe the effect of ITGβ6+exosomes in vitro. Elastic or stress-relaxing hydrogels based on Polymethyl Methacrylate (PMMA) and acrylic acid-WGG(KA)3-heparin were constructed to mimic the in situ ECM under normal and fibrotic conditions with different stiffness (2kPa, 50kPa). Exosomes in hydrogels were tracked by single particle tracking to investigate their mobility and retention capability. Purified exosomal integrin β6 and inhibitor of exosome secretion, GW4869 was applied to clarify the role of exosomal integrin in latent TGF-β1 activation. Results In UUO model, immunofluorescence staining showed that latent TGF-β1 localized to ECM space and stored in the renal tubular interstitial which was activated with fibrotic progression. Additionally, we observed integrin β6 expression in tubule or interstitial increased with fibrotic progression. Isolated exosomes derived from cultured tubule cells under serum-free condition or renal tissues of UUO mice showed higher proportion of integrin β6+ subpopulation, as demonstrated via single exosome analysis by Nanoimaging (Oxford) . Interestingly, these exosomes diffused through hydrogels, while maintaining their original morphology and particle size. It turned out that stress-relaxing hydrogels with lower stiffness exhibited better retention of exosomes. Then, transwell experiment and single-particle tracking both revealed increased exosome diffusion rate in ECM with higher stiffness. Due to the critical role of integrin in the activation of latent TGFβ, we propose integrin β6+ exosome may exert such effect in ECM microenvironment. Impressively, purified ITGB6+ exosomes significantly increased TGF-β1 activation in TGF-β1 overexpressed HK-2 cells, while inhibiting exosome secretion via GW4869 treatment reversed such activation remarkably. Conclusion We demonstrated that tubular epithelial cell derived exosomal integrin β6 diffused in ECM space and promoted the activation of latent TGF-β1. Higher ECM stiffness correlates with accelerated movement of exosome which enhanced TGFβ1 activation. Exosomal integrin β6 may represent a novel manner of TGF-β1 activation with signal-spreading potential which may become a new therapeutic target for suppression of TGF-β1 activation in CKD treatment.