Integrin Blockade Research Articles

TMIC-38. INTEGRIN BLOCKING PEPTIDE REPROGRAMS THE IMMUNOSUPPRESSIVE MICROENVIRONMENT OF EXPERIMENTAL GLIOMAS IMPROVING ANTI-PD-1 THERAPY OUTCOME

Abstract Clinical trials with immune checkpoint inhibitors (ICI) have benefited many cancer patients but failed in a number of tumors, including glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. The main obstacle for ICI efficacy in GBM is the continuously evolving tumor microenvironment (TME), in which antitumor immunity is inhibited or eluded by tumor-secreted factors. Accumulation and reprogramming of myeloid cells (GAMs) creates a “cold” immunosuppressive TME with a poor infiltration and exhaustion of effector T cells. We employ Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) with 40 protein markers along with spatial transcriptomics and immunophenotyping to dissect identities and functionalities of immune cells (CD45+) in experimental GL261 gliomas. The results revealed identities of immune cells instrumental for creating the immunosuppressive milieu and cell-cell communication networks. We have developed a designer RGD peptide that blocks the reprogramming of GAMs by targeting tumor-GAMs interactions. We demonstrate that RGD efficiently blocks microglia-dependent invasion of murine and human glioma cells in vitro. We explored if the intratumorally delivered RGD can revert tumor-induced changes in the TME of experimental gliomas and improve anti-PD-1 immunotherapy. While RGD alone did not reduce tumor growth in vivo, it prevented reprogramming of myeloid cells into protumoral GAMs and led to the normalization of peritumoral blood vessels. Combining RGD with anti-PD-1 antibody resulted in reduced tumor growth, increase in percentages of proliferating, interferon-ɣ producing CD8+T cells and depletion of regulatory T cells. Transcriptomic profiles of GAMs were altered by the combined treatment, consistently with the restored “hot” inflammatory TME which resulted in boosting immunotherapy responses. Intratumorally delivered RGD modified in the same way functionalities of GAMs in the TME of human U87MG gliomas in nude mice. Our results demonstrate that the integrin blockade combined with immune checkpoint inhibitors reinvigorates both innate and adaptive antitumor immunity. The results pave the way to improve responses to immunotherapy in GBM and other cancers.

Effect of Integrin Blockade on Experimental Spondyloarthritis

Spondyloarthritis (SpA) describes a group of diseases characterized by chronic inflammation in the spine and peripheral joints. While pathogenesis is still unclear, proinflammatory gut-derived immune cells have been identified in the joints of SpA patients. We previously identified an enriched population of integrin-expressing cells in the joints of SpA patients. Entry of gut-derived cells into joints may be mediated by these integrins. In the current study, we used the SKG murine model of SpA to study the impact of integrin blockade. Mice were injected with antibodies against the integrin α4β7 or the β7 monomer twice a week. Treatment with antibodies against α4β7 reduced disease severity in curdlan-injected SKG mice, with disease scores being comparable between treatment initiation times. Targeting the β7 monomer led to reduced arthritis severity compared to targeting the α4β7 dimer. Treatment with antibodies against α4β7 or β7 decreased expression of these integrins in CD4+ T cells, with the frequency of αE+β7+ T cells in the spleen and lymph nodes correlating with disease severity. In summary, we showed that integrin blockade showed potential for ameliorating disease in a murine model of SpA, lending support for further studies testing integrin blockade in SpA.

The Role of αvβ3 Integrin in Lamina Cribrosa Cell Mechanotransduction in Glaucoma.

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVβ3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVβ3 integrin expression using cilengitide (a known potent αVβ3 and αVβ5 inhibitor) and β3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVβ3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVβ3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVβ3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVβ3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVβ3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVβ3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVβ3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVβ3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma.

Dual targeting of αv and α5β1 integrins in triple-negative breast cancer (TNBC).

e15073 Background: Integrin αv has been implicated in a stem-phenotype of breast cancer (1) and is cross-regulated by fibronectin-binding integrin α5β1 in tumor cells (2). We hypothesized that these integrins are expressed in TNBC cells and dual targeting of αv and α5β1 integrins by a bispecific antibody (BsAbαv/α5β1) will be superior to monospecific integrin antibodies (MsAbs) alone or in combination. Methods: A representative panel of basal-subtype TNBC lines was assembled: MDA-MB-231 (P53 mutated, KRAS mutated), MDA-MB-468 (P53 mutated, PTEN deleted, EGFR amplified) and BT-20 (P53 and PIK3CA mutated, EGFR amplified). Integrin expression was determined by flow cytometry and Western blot in cell lines and by IHC in the residual cancer burden (RCB) of TNBC specimens following neoadjuvant chemotherapy (positive: &gt;2+ membranous, &gt;10% cells). The impact of MsAbs targeting αv and α5β1 integrins alone or in combination versus the BsAbαv/α5β1 was assessed in adhesion, migration, and clonogenic survival. The impact of integrin blockade on nuclear localization of basal markers including FAK, YAP, EGFR and beta-catenin, was assessed. Results: Robust integrin αv expression was present in all TNBC lines with variable α5β1 expression. Marked reduction in α5 and in αv expression following BsAbαv/α5β1 was uniquely demonstrated by Western blot and/or flow cytometry compared to MsAbs, likely due to endosomal trafficking and lysosomal degradation of target integrins as reported previously (Joshi, MCR. 2020). Combined α5 and αv neutralization with MsAbs was superior to individual single agents in blocking adhesion and migration across all TNBC lines studied. However, the BsAbα5β1/αv was superior to combinatorial MsAbs in MDA-MB-468 and BT-20 lines in chemotaxis assays. A significant (&gt;50%) reduction in clonogenic cell survival was noted with the BsAbα5β1/αv compared to MsAbs. BsAbαv/α5β1 blocked nuclear localization of FAK, EGFR, activated beta-catenin, and YAP in MDA-MB-231 uniquely compared to MsAbs alone or in combination. RCB tumor cells were strongly positive for membranous αv [7/9, (78%)]. Diffuse expression of integrin α5 (67%) in alpha smooth-muscle actin-expressing fibroblasts with dual staining for integrin αv was found. Marked CD68+ macrophage and CD3+ infiltration was seen in tumor and stroma of all RCB tumors. Conclusions: Combinatorial α5 and αv integrin targeting with BsAbα5/αvβ1 defines a novel therapeutic strategy with a distinct mechanism of action for further investigation in TNBC. Enrichment of integrins αv and α5 in the inflamed RCB of TNBC tumors and stroma respectively point to a potential role of these integrins in epithelial-stromal interactions and therapeutic resistance. 1. Desgrosellier, Dev Cell. 2014. 2. Joshi, MCR. 2020.

Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbβ3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s−1) and low (300s−1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

Abstract 6575: Selective targeting of integrins αVβ8 and αVβ1 within the dynamic ecosystem of pancreatic cancer to improve the overall anti-tumor response

Abstract Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival of less than 10% and remains the 3rd leading cause of cancer-related death in Western societies. New treatment options are urgently needed. TGF-β promotes stromal cell reprogramming, immunosuppression, and fibrinogenesis in cancers, including PDA. Integrins αVβ8 and αVβ1 are important activators of TGF-β signalling. Selective integrin blockade has recently emerged as a promising therapeutic approach to address TGF-β-mediated immunotherapy resistance and improve anti-tumour response across cancer models. The purpose of this study was to assess the in vivo efficacy of small molecule inhibitor PLN-104, a selective inhibitor of αVβ1, and PLN-101095, a dual inhibitor of αVβ8 and αVβ1, in well-annotated models of advanced PDA. We determined the preclinical efficacy and detailed anti-stromal effect of PLN-101095 inhibitor in genetically defined and patient-derived PDA models, including clinically relevant combinations with standard of care (SoC) chemotherapy and anti-programmed death receptor-1 antibody (anti-mPD-1). Mechanistic assessment of alterations in tumour cell-stromal cell crosstalk was performed using comprehensive transcriptomics and immunofluorescence approaches. Dual targeting of αVβ8 and αVβ1 with PLN-101095 in the syngeneic LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre (KPC) model effectively reduced tumour growth by 45% in comparison to the vehicle, and significantly delayed disease progression in vivo. Single-cell analysis of PLN-101095-treated KPC pancreatic tumours revealed a positive reprogramming of malignant cells from a mesenchymal to a more epithelial-like state. This effect was further associated with an upregulation of MHC type I/II markers and corresponding downregulation of pro-metastatic factors across diverse cancer sub-populations. Additionally, combining PLN-101095 with anti-mPD-1 antibody further improved survival in this aggressive model of metastatic PDA. In a second syngeneic model, Pan02, PLN-101095 in combination with anti-mPD-1 antibody significantly reduced tumour growth, while increasing CD8+ lymphocyte infiltration. The combination of PLN-101095 with Anti-PD1 increased MHC and IFN gene expression. Finally, utilizing patient-derived models of metastatic PDA revealed that both PLN-104 and PLN-101095 significantly blocked tumour growth, improved the response to SoC chemotherapy Gemcitabine/Abraxane, and reduced metastatic spread to distant sites. In conclusion, these data provide scientific rationale for the design of future PLN-101095 and SoC chemotherapy as well as immunotherapy combinations in pancreatic cancer, with Phase I first-in-human oncology studies with PLN-101095 plus ICB already underway. Citation Format: Dannielle Upton, Diego Chacon Fajardo, Sofia Omari, Sean Porazinski, Benjamin McLean, Diana Schuhmacher, Aji Istadi, Australian Pancreatic Cancer Genome Initiative, Vishal Kothari, Darren Finkelstein, Tim Machajewski, Fernando Rock, Scott Turner, Marina Pajic. Selective targeting of integrins αVβ8 and αVβ1 within the dynamic ecosystem of pancreatic cancer to improve the overall anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6575.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the β5 integrin

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvβ5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin β5 complexes with the insulin receptor beta (IRβ) in skeletal muscle, resulting in dephosphorylation of IRβ and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between β5 and IRβ impacts IRβ phosphorylation status. We show in invitro and invivo in skeletal muscle in mice that antibody-mediated blockade of the β5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRβ resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The β5-PTP1B complex is recruited by MFGE8 to IRβ leading to termination of canonical insulin signaling. β5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and β5 in regulating insulin signaling.

Chemerin triggers migration of a CD8 T cell subset with natural killer cell functions

The recruitment of cells with effector functions into the tumor microenvironment holds potential for delaying cancer progression. We show that subsets of human CD28-effector CD8 Tcells, CCR7- CD45RO+ effector memory, and CCR7- CD45RO- effector memory RA phenotypes, express the chemerin receptor CMKLR1 and bind chemerin via the receptor. CMKLR1-expressing human CD8 effector memory Tcells present gene, protein, and cytotoxic features of NK cells. Active chemerin promotes chemotaxis of CMKLR1-expressing CD8 effector memory cells and triggers activation of the α4β1 integrin. In an experimental prostate tumor mouse model, chemerin expression is downregulated in the tumor microenvironment, which is associated with few tumor-infiltrating CD8+ Tcells, while forced overexpression of chemerin by mouse prostate cancer cells leads to an accumulation of intra-tumor CD8+ Tcells. Furthermore, α4 integrin blockade abrogated the chemerin-dependent recruitment of CD8+ T effector memory cells into implanted prostate tumors invivo. The results identify a role for chemerin:CMKLR1 in defining a specialized NK-like CD8 Tcell, and suggest the use of chemerin-dependent modalities to target effector CMKLR1-expressing Tcells to the tumor microenvironment for immunotherapeutic purposes.

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway.

Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5β1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of β1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased β1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or β1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and β1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

β1 integrin is essential for blood–brain barrier integrity under stable and vascular remodelling conditions; effects differ with age

BackgroundMaintaining a tight blood–brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard.MethodsHere, in young (8–10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking β1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test.ResultsIn both young and aged mice, β1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, β1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, β1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, β1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that β1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins.ConclusionsThese data demonstrate that β1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As β1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing β1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

β1-Integrin blockade prevents podocyte injury in experimental models of minimal change disease

IntroductionActivation of the focal adhesion kinase (FAK) in podocytes is involved in the pathogenesis of minimal change disease (MCD), but the pathway leading to its activation in this disease is unknown. Here, we tested whether podocyte β1 integrin is the upstream modulator of FAK activation and podocyte injury in experimental models of MCD-like injury. MethodsWe used lipopolysaccharide (LPS) and MCD sera to induce MCD-like changes in vivo and in cultured human podocytes, respectively. We performed functional studies using specific β1 integrin inhibitors in vivo and in vitro, and integrated histological analysis, western blotting, and immunofluorescence to assess for morphological and molecular changes in podocytes. By ELISA, we measured serum LPS levels in 35 children with MCD or presumed MCD (idiopathic nephrotic syndrome [INS]) and in 18 healthy controls. ResultsLPS-injected mice showed morphological (foot process effacement, and normal appearing glomeruli on light microscopy) and molecular features (synaptopodin loss, nephrin mislocalization, FAK phosphorylation) characteristic of human MCD. Administration of a β1 integrin inhibitor to mice abrogated FAK phosphorylation, and ameliorated proteinuria and podocyte injury following LPS. Children with MCD/INS in relapse had higher serum LPS levels than controls. In cultured human podocytes, β1 integrin blockade prevented cytoskeletal rearrangements following exposure to MCD sera in relapse. ConclusionsPodocyte β1 integrin activation is an upstream mediator of FAK phosphorylation and podocyte injury in models of MCD-like injury.

Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.

β1-Integrin blockade prevents podocyte injury in experimental models of minimal change disease

IntroductionActivation of the focal adhesion kinase (FAK) in podocytes is involved in the pathogenesis of minimal change disease (MCD), but the pathway leading to its activation in this disease is unknown. Here, we tested whether podocyte β1 integrin is the upstream modulator of FAK activation and podocyte injury in experimental models of MCD-like injury. MethodsWe used lipopolysaccharide (LPS) and MCD sera to induce MCD-like changes in vivo and in cultured human podocytes, respectively. We performed functional studies using specific β1 integrin inhibitors in vivo and in vitro, and integrated histological analysis, western blotting, and immunofluorescence to assess for morphological and molecular changes in podocytes. By ELISA, we measured serum LPS levels in 35 children with MCD or presumed MCD (idiopathic nephrotic syndrome [INS]) and in 18 healthy controls. ResultsLPS-injected mice showed morphological (foot process effacement, and normal appearing glomeruli on light microscopy) and molecular features (synaptopodin loss, nephrin mislocalization, FAK phosphorylation) characteristic of human MCD. Administration of a β1 integrin inhibitor to mice abrogated FAK phosphorylation, and ameliorated proteinuria and podocyte injury following LPS. Children with MCD/INS in relapse had higher serum LPS levels than controls. In cultured human podocytes, β1 integrin blockade prevented cytoskeletal rearrangements following exposure to MCD sera in relapse. ConclusionsPodocyte β1 integrin activation is an upstream mediator of FAK phosphorylation and podocyte injury in models of MCD-like injury.

The actin cytoskeleton-MRTF/SRF cascade transduces cellular physical niche cues to entrain the circadian clock.

The circadian clock is entrained to daily environmental cues. Integrin-linked signaling via actin cytoskeleton dynamics transduces physical niche cues from the extracellular matrix to myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated transcription. The actin cytoskeleton organization and SRF-MRTF activity display diurnal oscillations. By interrogating disparate upstream events in the actin cytoskeleton-MRTF-A/SRF signaling cascade, we show that this pathway transduces extracellular niche cues to modulate circadian clock function. Pharmacological inhibition of MRTF-A/SRF by disrupting actin polymerization or blocking the ROCK kinase induced period lengthening with augmented clock amplitude, and genetic loss of function of Srf or Mrtfa mimicked the effects of treatment with actin-depolymerizing agents. In contrast, actin polymerization shortened circadian clock period and attenuated clock amplitude. Moreover, interfering with the cell-matrix interaction through blockade of integrin, inhibition of focal adhesion kinase (FAK, encoded by Ptk2) or attenuating matrix rigidity reduced the period length while enhancing amplitude. Mechanistically, we identified that the core clock repressors Per2, Nr1d1 and Nfil3 are direct transcriptional targets of MRTF-A/SRF in mediating actin dynamics-induced clock response. Collectively, our findings defined an integrin-actin cytoskeleton-MRTF/SRF pathway in linking clock entrainment with extracellular cues that might facilitate cellular adaptation to the physical niche environment.

Human intestinal myofibroblasts deposited collagen VI enhances adhesiveness for T cells – A novel mechanism for maintenance of intestinal inflammation

Objective: Inflammatory bowel diseases (IBD) cause chronic intestinal damage and extracellular matrix (ECM) remodeling. The ECM may play an active role in inflammation by modulating immune cell functions, including cell adhesion, but this hypothesis has not been tested in IBD.Design: Primary human intestinal myofibroblast (HIMF)-derived ECM from IBD and controls, 3D decellularized colon or ECM molecule-coated scaffolds were tested for their adhesiveness for T cells. Matrisome was analysed via proteomics. Functional integrin blockade was used to investigate the underlying mechanism. Analysis of the pediatric Crohn's disease (CD) RISK inception cohort was used to explore an altered ECM gene expression as a potential predictor for a future complicated disease course.Results: HIMF-derived ECM and 3D decellularized colonic ECM from IBD bound more T cells compared to control. Control HIMFs exposed to the pro-inflammatory cytokines Iinterleukin-1β (IL-1β) and tumor necrosis factor (TNF) increased, and to transforming growth factor-β1 (TGF-β1) decreased ECM adhesiveness to T cells. Matrisome analysis of the HIMF-derived ECM revealed collagen VI as a major culprit for differences in T cell adhesion. Collagen VI knockdown in HIMF reduced adhesion T cell as did the blockage of integrin αvβ1. Elevated gene expression of collagen VI in biopsies of pediatric CD patients was linked to risk for future stricturing disease.Conclusion: HIMF-derived ECM in IBD binds a remarkably enhanced number of T cells, which is dependent on Collagen VI and integrin αvβ1. Collagen VI expression is a risk factor for a future complicated CD course. Blocking immune cells retention may represent a novel approach to treatment in IBD.

Adventitia-derived extracellular matrix hydrogel enhances contractility of human vasa vasorum-derived pericytes via α2 β1 integrin and TGFβ receptor.

Pericytes are essential components of small blood vessels and are found in human aortic vasa vasorum. Prior work uncovered lower vasa vasorum density and decreased levels of pro-angiogenic growth factors in adventitial specimens of human ascending thoracic aortic aneurysm. We hypothesized that adventitial extracellular matrix (ECM) from normal aorta promotes pericyte function by increasing pericyte contractile function through mechanisms deficient in ECM derived from aneurysmal aortic adventitia. ECM biomaterials were prepared as lyophilized particulates from decellularized adventitial specimens of human and porcine aorta. Immortalized human aortic adventitia-derived pericytes were cultured within Type I collagen gels in the presence or absence of human or porcine adventitial ECMs. Cell contractility index was quantified by measuring the gel area immediately following gelation and after 48 h of culture. Normal human and porcine adventitial ECM increased contractility of pericytes when compared with pericytes cultured in absence of adventitial ECM. In contrast, aneurysm-derived human adventitial ECM failed to promote pericyte contractility. Pharmacological inhibition of TGFβR1 and antibody blockade of α2 β1 integrin independently decreased porcine adventitial ECM-induced pericyte contractility. By increasing pericyte contractility, adventitial ECM may improve microvascular function and thus represents a candidate biomaterial for less invasive and preventative treatment of human ascending aortic disease.

Pharmacological blockade of αV integrin (CD51) reduces the development of pressure-overload-induced cardiac fibrosis

Myocardial fibrosis is involved in most forms of heart diseases, progressively leading to adverse cardiac remodeling and heart failure. We previously identified a population of cardiac stromal cells that contribute to fibrosis development through a CD51 (alpha v integrin) dependent activation of pro-fibrotic pathways in a murine model of myocardial infarction. The pharmacological blockade of CD51 was associated with a significant reduction of fibrosis, preservation of cardiac function and improved survival. Investigate the impact of CD51 pharmacological blockers on the development of cardiac fibrosis induced by neurohormonal pro-hypertrophic stressors. We used C57Bl/6J mice infused with Angiotensin II (1.44 mg/kg/day) or dual Angiotensin II (1.44 mg/kg/day) + PhenylEphrine (50 mg/kg/day) through an osmotic pump for 28 or 14 days, respectively. Animals were treated with daily with intra-peritoneal injection of either CD51 blocker cilengitide or vehicle for the duration of the study. We found that both AngII and AngII + PE stimulation led to a similar cardiac hypertrophic remodeling: heart weight on body weight ratio as well as cardiomyocyte size were similarly increased. However, AngII + PE mice developed a significantly higher amount of interstitial fibrosis as compared to AngII mice (10.2 ± 4.3% vs. 4.5 ± 1.3% of total area respectively, P < 0.0001). Importantly, AngII + PE but not AngII mice exhibited a significant increase in CD51 expression and in the number of CD51+ cells as found in cytometry analysis. CD51 blockade significantly decreased the development of fibrosis under AngII + PE stimulation (7.6% ± 1.7, P < 0.05), as well as heart weight on body weight ratio (5.4 ± 0.5 vs. 5.7 ± 0.4, P < 0.05), but did not change the cardiomyocyte sizes. CD51 blockade also partially rescued the progressive drop in LVEF observed in mice stimulated with AngII + PE (47.5% ± 4.0 vs. 40.7% ± 13.8). Further cytometry analyses on cardiac tissues demonstrated that CD51 is expressed by stromal cells and by CCR2+ macrophages. Preliminary results indicate that the anti-fibrotic effects of CD51 blockade could come from a dual reduction on the activation of stromal and the immune-inflammatory response. Inhibition of CD51 reduces the development of cardiac fibrosis in response to pressure-overload, through the targeting of both stromal and inflammatory cardiac cells.

Blockade of integrin signaling reduces chemotherapy-induced premature senescence in collagen cultured bladder cancer cells.

BackgroundDiminished sensitivity towards chemotherapy remains the major impediment to the clinical treatment of bladder cancer. However, the critical elements in control of chemotherapy resistance remain obscure.MethodsWe adopted improved collagen gels and performed cytotoxicity analysis of doxorubicin (DOX) and mitomycin C (MMC) of bladder cancer cells in a 3D culture system. We then detected the expression of multidrug resistant gene ABCB1, dormancy-associated functional protein chicken ovalbumin upstream-transcription factor 1 (COUPTF1), cell proliferation marker Ki-67, and cellular senescence marker senescence-associated β-galactosidase (SA-β-Gal) in these cells. We further tested the effects of integrin blockade or protein kinase B (AKT) inhibitor on the senescent state of bladder cancer. Also, we examined the tumor growth and survival time of bladder cancer mouse models given the combination treatment of chemotherapeutic agents and integrin α2β1 ligand peptide TFA (TFA).ResultsCollagen gels played a repressive role in bladder cancer cell apoptosis induced by DOX and MMC. In mechanism, collagen activated the integrin β1/AKT cascade to drive bladder cancer cells into a premature senescence state via the p21/p53 pathway, thus attenuating chemotherapy-induced apoptosis. In addition, TFA had the ability to mediate the switch from senescence to apoptosis of bladder cancer cells in xenograft mice. Meanwhile, TFA combined with chemotherapeutic drugs produced a substantial suppression of tumor growth as well as an extension of survival time in vivo.ConclusionsBased on our finding that integrin β1/AKT acted primarily to impart premature senescence to bladder cancer cells cultured in collagen gel, we suggest that integrin β1 might be a feasible target for bladder cancer eradication.

CCN2-induced lymphangiogenesis is mediated by the integrin \u03b1v\u03b25\u2013ERK pathway and regulated by DUSP6

Lymphangiogenesis is essential for the development of the lymphatic system and is important for physiological processes such as homeostasis, metabolism and immunity. Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated the effects of CCN2 on lymphangiogenesis. In in vivo Matrigel plug assays, exogenous CCN2 increased the number of Podoplanin-positive vessels. Subsequently, we found that CCN2 induced phosphorylation of ERK in primary cultured LECs, which was almost completely inhibited by the blockade of integrin αvβ5 and partially decreased by the blockade of integrin αvβ3. CCN2 promoted direct binding of ERK to dual-specific phosphatase 6 (DUSP6), which regulated the activation of excess ERK by dephosphorylating ERK. In vitro, CCN2 promoted tube formation in LECs, while suppression of Dusp6 further increased tube formation. In vivo, immunohistochemistry also detected ERK phosphorylation and DUSP6 expression in Podoplanin-positive cells on CCN2-supplemented Matrigel. These results indicated that CCN2 promotes lymphangiogenesis by enhancing integrin αvβ5-mediated phosphorylation of ERK and demonstrated that DUSP6 is a negative regulator of excessive lymphangiogenesis by CCN2.

Insulin-like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism.

Simple SummaryInsulin-like growth factor-1 (IGF-1) is a growth hormone and is implicated in prostate cancer progression. Most prostate cancers begin in an androgen-dependent state so that androgen deprivation therapy results in improved clinical outcome. However, some cancerous cells may survive androgen deprivation, growing into therapy-resistant, androgen-independent prostate cancer. The present study investigated the influence of IGF-1 on tumor growth and migration properties using androgen-dependent LNCaP and VCaP and androgen-independent PC3 and DU145 prostate cancer cells. Stimulation with IGF-1 activated growth in all cell lines. There were changes in transmembrane receptors (integrins) that bind cells to each other and changes in focal adhesion kinase that controls cell motility. Intracellular Akt/mTOR signaling, regulating cell division, was also activated. Thus, it seems that prostate cancer progression is controlled by a fine-tuned network between IGF-1-driven integrin-FAK signaling and the Akt-mTOR pathway. Concerted targeting of both pathways may, therefore, help prevent cancer dissemination.Insulin-like growth factor-1 (IGF-1)-related signaling is associated with prostate cancer progression. Links were explored between IGF-1 and expression of integrin adhesion receptors to evaluate relevance for growth and migration. Androgen-resistant PC3 and DU145 and androgen-sensitive LNCaP and VCaP prostate cancer cells were stimulated with IGF-1 and tumor growth (all cell lines), adhesion and chemotaxis (PC3, DU145) were determined. Evaluation of Akt/mTOR-related proteins, focal adhesion kinase (FAK) and integrin α and β subtype expression followed. Akt knock-down was used to investigate its influence on integrin expression, while FAK blockade served to evaluate its influence on mTOR signaling. Integrin knock-down served to investigate its influence on tumor growth and chemotaxis. Stimulation with IGF-1 activated growth in PC3, DU145, and VCaP cells, and altered adhesion and chemotactic properties of DU145 and PC3 cells. This was associated with time-dependent alterations of the integrins α3, α5, αV, and β1, FAK phosphorylation and Akt/mTOR signaling. Integrin blockade or integrin knock-down in DU145 and PC3 cells altered tumor growth, adhesion, and chemotaxis. Akt knock-down (DU145 cells) cancelled the effect of IGF-1 on α3, α5, and αV integrins, whereas FAK blockade cancelled the effect of IGF-1 on mTOR signaling (DU145 cells). Prostate cancer growth and invasion are thus controlled by a fine-tuned network between IGF-1 driven integrin-FAK signaling and the Akt-mTOR pathway. Concerted targeting of integrin subtypes along with Akt-mTOR signaling could, therefore, open options to prevent progressive dissemination of prostate cancer.