Macrophage Cell Migration Research Articles

Abstract 7628: Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy

Abstract Metastatic disease drastically reduces clinical prognosis, and remains the primary cause of mortality from solid cancers, such as breast and pancreatic cancer. Since targeted therapeutic options are limited, we developed small molecule compounds to block activation (GTP loading) of the Rho GTPases Rac and Cdc42. Rac and Cdc42 are ideal molecular targets due to their regulation of cell migration, proliferation, invasion, and signaling in cancer and immunosuppressive cells in the tumor microenvironment. Our lead compound MBQ-167 is a dual Rac and Cdc42 inhibitor with IC50 of ~100 nM for inhibition of Rac/Cdc42 activation and the phosphorylation of their common downstream effector p21-activated kinase (PAK). MBQ-167 is highly effective in mouse models of breast cancer metastasis and is currently undergoing a Phase I clinical trial in advanced breast cancer patients. The purpose of this study is to investigate potential target molecules that can be used as biomarkers predictive of Rac/Cdc42 inhibitor efficacy. We recently published that in addition to inhibiting the growth and migration of breast and pancreatic cancer cells, MBQ-167 inhibits macrophage cell migration, immunosuppressive myeloid cell activation and release of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cancer. Transcriptomic data of tumors from mice treated with vehicle or MBQ-167 identified a number of immunomodulatory pathways downregulated by MBQ-167, including IL-6 and S100A family signaling, and Chitinase 3-like (CHI3L1/CHI4L1) genes coding for the human YKL-40 protein. We tested for the activation status of PAK (phospho (p)-PAK) as a diagnostic marker for Rac/Cdc42 inhibitor efficacy using breast cancer patient tissue in ex vivo culture treated with vehicle or MBQ-167 and found decreased p-PAK staining following immunohistochemistry of MBQ-167-treated tissue. From flow cytometry, using Alexa-488-tagged anti p-PAK antibody, we found that p-PAK was reduced from peripheral blood mononuclear cells (PBMCs) following MBQ-167. ELISA assays for YKL-40/CHI3L1 proteins after treatment of pancreatic cancer cell lines and macrophage-like cells with Rac/Cdc42 inhibitors show that MBQ-167 and the derivative MBQ-168 reduced CHI3L1 levels from conditioned media. These results demonstrate the potential of p-PAK and YKL-40/CHI3L1 from blood as biomarkers for Rac/Cdc42 inhibitor efficacy. This study impacts pharmacodynamic biomarker design for the planned Phase 2 clinical trials for MBQ-167 in breast and pancreatic cancer. Citation Format: Anamaris Torres-Sanchez, Ailed M. Cruz-Collazo, Michael Rivera-Robles, Olga Katsara, Stephanie Dorta-Estremera, Viviana Negron, Victor P. Carlo, Robert J. Schneider, Suranganie Dharmawardhane. Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy [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 7628.

SPI1‐related protein inhibits cervical cancer cell progression and prevents macrophage cell migration

The functions and molecular mechanisms of SPI1-related protein (SPIB) were examined in cervical cancer (CC) cells. Genes related to miscarriage and prognosis in CC were identified by Kaplan-Meier and differential expression analysis, respectively. Cell proliferation, apoptosis, migration, and invasion were examined by cell counting kit-8, flow cytometry, transwell migration, and transwell invasion assays, respectively. The potential functions and molecular mechanisms of SPIB in CC were speculated by gene set enrichment analysis (GSEA) analysis. The mRNA and protein levels of genes were examined by RT-qPCR and western blot assays, respectively. The effect of SPIB on macrophage cells was tested by macrophage recruitment assay and bioinformatics analysis. A total of 753 dysregulated genes were identified in 88 TCGA CC samples with a history of one or more miscarriages versus 208 CC samples with no miscarriage history. Also, 91 genes related to CC prognosis were identified. SPIB, a gene related to both miscarriage and CC prognosis, inhibited Hela cell proliferation, migration, and invasion, and facilitated Hela cell apoptosis. GSEA analysis disclosed that SPIB might play vital roles in immunity, chemokine signaling pathway, and macrophage chemotaxis/activation in CC. Moreover, SPIB inhibited C-X-C motif chemokine ligand 8 (CXCL8), C-C motif chemokine ligand 17 (CCL17), and C-C motif chemokine ligand 25 (CCL25) expression in Hela cells, and SPIB overexpression in Hela cells hampered THP-1 cell migration. Higher SPIB expression was associated with less M2 macrophage infiltration in CC. SPIB inhibited CC-cell progression and hindered macrophage cell migration in CC.

Effects of Netrin-1 and NHE1 Participation on the Migration of Macrophages Driven by CCL2.

This experiment was designed to investigate the relationship between NHE1 gene expression differences between Netrin-1 and NHE1. For this purpose, the blank control, CCL2, CCL2 + Netrin-1 groups were constructed, and cell migration ability was detected by scratch tests and Transwell experiments; Commercial over-expressed NHE1 adenovirus vector (over-expressed NHE1 group), shRNA adenoviral vector silencing NHE1 (silencing NHE1 group) and negative control without carrying virus (negative control group) were subjected to RT-PCR test 24h after infection and pH recovery rate after acid loading was measured. The percentage of wound healing area and the number of cell migration of macrophages in the blank control group, CCL2 group, CCL2+Netrin-1 group, over-expressed NHE1 group, silencing NHE1 group and negative control group were compared. Results showed that in terms of migration ability, the percentage of wound healing area and migration in CCL2 increased (P <0.05), in CCL2 + Netrin-1 (P <0.05) and increased NHE1 mRNA (P <0.05), and not in NHE1 (P <0.05).pH response rate after acid load (NHE1 activity) showed that NHE1 activity was enhanced compared with the blank group, while NHE1 activity in silent NHE1 group decreased (P <0.05); from macrophage migration ability after overexpression/silencing, the percentage of macrophage wound healing area and cell migration increased/decreased compared with CCL2 group and Netrin-1 + CCL2 group (P <0.05). Then Upregulation of NHE1 can promote CCL2-driven macrophage RAW264.7 cell migration, and the downregulation of NHE1 can inhibit its cell migration; Netrin-1 can inhibit CCL2-driven RAW264.7 cell migration regardless of NHE1 regulation.

Rac and Cdc42 Inhibitors in Rheumatoid Arthritis Therapy

Rheumatoid Arthritis (RA) is the most common autoimmune disorder characterized by chronic inflammation due to synovial fibroblast proliferation, neovascularization, leukocyte extravasation, and progressive joint destruction. Most RA medications focus on relieving symptoms of chronic inflammation, rather than prevent the inflammatory cells from attacking the joints and the underlying cartilage and bone. These initial processes have been shown to be under the regulation of key signaling proteins Rac and Cdc42. Therefore, the objective of this study was to demonstrate the effect of our patented Rac/Cdc42 inhibitors MBQ‐167 and EHop‐016 in inflammatory cells in vitro. Our hypothesis is that inflammatory cells in the synovium can be inhibited by Rac/Cdc42 inhibitors that have the potential as a RA therapeutics. To test this hypothesis, macrophage‐like cell lines WBC264‐9C human leukocytes or THP1 human macrophage were incubated with 0‐500 nM of MBQ‐167, lysed and subjected to Rac.GTP pulldown assays. Results showed that Rac activation was inhibited by 46‐56% at 250 nM and by 60‐77% at 500 nM MBQ‐167 for both cell lines. Total Rac levels were not significantly changed by MBQ‐167 in concentrations up to 500 nM. Moreover, preliminary results on synovial fibroblasts SW‐982 demonstrated inhibition of Rac activation in response to MBQ‐167 at 750 nM or higher. Next, macrophage‐like cells were treated with the Rac/Cdc42 inhibitors and tested for cell viability using a MTT assay. Results showed no effects on cell proliferation at high µM concentrations of Rac/Cdc42 inhibitors. Since Rac activation regulates cell migration, which is central to the inflammatory response in synovial joints, we tested the effect of MBQ‐167 in a wound healing assay. Incubation with MBQ‐167 at 250 or 500 nM inhibited macrophage migration. At 24h, 500 nM MBQ‐167 reduced macrophage cell migration by 80%. In conclusion, this data demonstrates that our Rac/Cdc42 inhibitors may be used to inhibit inflammatory cell action and their migration at sites of inflammation in RA, without affecting macrophage numbers, indicating no toxic effects in the immune system.

MiR-483-3p exacerbates pediatric pneumonia by suppressing IGF1 expression in alveolar macrophage

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the action of miR-483-3p in pediatric pneumonia and identify potential biomarkers.&#x0D; Methods: Bronchoalveolar lavage was collected from 38 pneumonia patients and 25 healthy children. The expression of miR-483-3p in the lavage was determined using qRT-PCR. Alveolar macrophages collected from lavage of healthy children were cultured and used for functional assays. Transwell assay was conducted to evaluate macrophage cell migration. Cell viability and apoptosis were evaluated in lipopolysaccharide (LPS)-induced human pulmonary alveolar epithelial cells (HPAEpiCs) by CCK8 (cell counting kit - 8) or flow cytometry, respectively.&#x0D; Results: MiR-483-3p was significantly elevated in bronchoalveolar lavage of pneumonia patients, when compared to healthy children (p &lt; 0.001). MiR-483-3p, which targets insulin-like growth factor 1 (IGF1), decreased the mRNA and protein expression of IGF1 in alveolar macrophages collected from the lavage of healthy children. MiR-483-3p reduced motility of macrophages. IGF1 counteracted the LPS- induced decrease in cell viability and the increase in apoptosis of HPAEpiCs. Conditioned medium from macrophages transfected with miR-483-3p inhibitor increased cell viability and reduced cellular apoptosis of LPS-induced HPAEpiCs. However, conditioned medium from macrophages transfected with miR-483-3p mimics decreased cell viability and increased apoptosis.&#x0D; Conclusion: MiR-483-3p negatively regulates IGF1 to promote progression of pediatric pneumonia, providing a potential therapeutic target in pediatric pneumonia.&#x0D; &#x0D; &#x0D; &#x0D;

HIV-1 uses dynamic podosomes for entry into macrophages.

Macrophages are one of the major targets of Human Immunodeficiency virus 1 (HIV-1) and play crucial roles in viral dissemination and persistence during AIDS progression. Here, we reveal the dynamic podosome-mediated entry of HIV-1 into macrophages. Inhibition of podosomes prevented HIV-1 entry into macrophages, while stimulation of podosome formation promoted viral entry. Single-virus tracking revealed the temporal and spatial mechanism of the dynamic podosome-mediated viral entry process. The core and ring structures of podosomes played complex roles in viral entry. The HIV coreceptor, CCR5, was recruited to form specific clusters at the podosome ring, where it participated in viral entry. The podosome facilitated HIV-1 entry with a rotation mode triggered by dynamic actin. Our discovery of this novel HIV-1 entry route into macrophages, mediated by podosomes critical for cell migration and tissue infiltration, provides a new view of HIV infection and pathogenesis, which may assist in the development of new antiviral strategies.IMPORTANCEMacrophages are motile leukocytes and play critical roles in HIV-1 infection and AIDS progression. Podosomes, as small dynamic adhesion microdomains driven by the dynamic actin cytoskeleton, are mainly involved in cell migration of macrophages. Herein, we found that HIV-1 uses dynamic podosomes to facilitate its entry into macrophages. Single-virus imaging coupled with drug assays revealed the mechanism underlying the podosome-mediated route of HIV-1 entry into macrophages, including the dynamic relationship between the viral particles and the podosome core and ring structures, the CCR5 coreceptor. The dynamic podosome-mediated entry of HIV-1 into macrophages will be very significant for HIV-1 pathogenesis, especially for viral dissemination via macrophage migration and tissue infiltration. Thus, we report a novel HIV-1 entry route into macrophages mediated by podosomes, which extends our understanding of HIV infection and pathogenesis.

A Novel Function of TLR2 and MyD88 in the Regulation of Leukocyte Cell Migration Behavior During Wounding in Zebrafish Larvae.

Toll-like receptor (TLR) signaling via myeloid differentiation factor 88 protein (MyD88) has been indicated to be involved in the response to wounding. It remains unknown whether the putative role of MyD88 in wounding responses is due to a control of leukocyte cell migration. The aim of this study was to explore in vivo whether TLR2 and MyD88 are involved in modulating neutrophil and macrophage cell migration behavior upon zebrafish larval tail wounding. Live cell imaging of tail-wounded larvae was performed in tlr2 and myd88 mutants and their corresponding wild type siblings. In order to visualize cell migration following tissue damage, we constructed double transgenic lines with fluorescent markers for macrophages and neutrophils in all mutant and sibling zebrafish lines. Three days post fertilization (dpf), tail-wounded larvae were studied using confocal laser scanning microscopy (CLSM) to quantify the number of recruited cells at the wounding area. We found that in both tlr2–/– and myd88–/– groups the recruited neutrophil and macrophage numbers are decreased compared to their wild type sibling controls. Through analyses of neutrophil and macrophage migration patterns, we demonstrated that both tlr2 and myd88 control the migration direction of distant neutrophils upon wounding. Furthermore, in both the tlr2 and the myd88 mutants, macrophages migrated more slowly toward the wound edge. Taken together, our findings show that tlr2 and myd88 are involved in responses to tail wounding by regulating the behavior and speed of leukocyte migration in vivo.

TMIC-51. GBP1 RECRUITS MACROPHAGES TO PROMOTE GLIOBLASTOMA GROWTH

Abstract Guanylate binding protein 1 (GBP1) is an interferon-inducible large GTPase which plays a key role in tumor development, but the molecular mechanism is poorly understood. Here we investigated whether GBP1 could influence the tumor microenvironment in glioblastoma, the most common and malignant brain tumor. We found that forced expression of GBP1 in glioblastoma cells induced macrophage polarization toward an M2 phenotype via upregulating Chemokine (C-C motif) ligand 2 (CCL2). CCL2 acted via its receptor C-C chemokine receptor 2 (CCR2) to enhance macrophage cell migration in vitro. The M2 macrophages in turn promoted glioblastoma cell proliferation and migration. The orthotopic mouse model showed that GBP1 recruited M2 macrophages into tumor to promote glioblastoma progression, and targeting CCL2/CCR2 signaling axis with a small molecule inhibitor RS504393 led to decreased macrophage attraction and M2 polarization and a significant tumor growth retardation and prolonged survival of tumor-bearing mice. Clinically, GBP1 expression positively correlated with M2 macrophage numbers and CCL2 expression in glioblastoma. Taken together, our results reveal that GBP1 modulates the tumor immune microenvironment through CCL2 induction to promote glioblastoma infiltrating growth, and targeting tumor-associated macrophages may represent a new therapeutic strategy against glioblastoma.

Impact of diet‐derived polyphenol EGCG on neurotensin response upon HL‐60 promyelocytic leukemia cells acquisition of a macrophage‐like phenotype

BackgroundThe activation of a neurotensin (NTS)/neurotensin receptor (NTSR) axis in T lymphocytes, macrophages, and dendritic cells suggests that NTS could modulate immune response and trigger prosurvival signaling in malignant human B lymphocytes. Such axis can therefore represent a potential therapeutic target in chronic B cell leukemia.MethodsIn this study, analysis of NTS and NTSR1‐3 expression in acute myeloid leukemia (AML) patients using Kaplan‐Meyer plots specifically highlighted NTS and NTSRs as poor clinical prognosis biomarkers. NTSRs gene and protein expression was also explored in undifferentiated and upon phorbol 12‐myristate 13‐acetate (PMA)‐mediated differentiation of HL‐60 suspension cells, an acute promyelocytic leukemia subset cell line model from AML, into CD11b+/CD14− adherent macrophages.ResultsWe found that high NTS internalization correlated with increased matrix metalloproteinase‐9 expression, and basal macrophage cell migration. Whereas NTS dose‐dependently enhanced undifferentiated HL‐60 cells chemotaxis, it rather inhibited cell migration in differentiated macrophages through decreased Erk, MEK and Src signaling pathways. Epigallocatechin‐3‐gallate (EGCG), a diet‐derived polyphenol with anti‐inflammatory and chemopreventive properties, prevented PMA‐induced CD11b expression and NTS internalization.ConclusionsOur study evidences specific regulation and role for NTS receptors in HL‐60 differentiated macrophages, which could in part account for their contribution to immune evasion or tumor‐associated inflammation, and be prevented by the anti‐cancer property of diet‐derived polyphenols.Support or Funding InformationFunding: UQAM Foundation and Chaire in Cancer Prevention and TreatmentThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Estramustine Phosphate Inhibits TGF-β-Induced Mouse Macrophage Migration and Urokinase-Type Plasminogen Activator Production.

Transforming growth factor-beta (TGF-β) has been demonstrated as a key regulator of immune responses including monocyte/macrophage functions. TGF-β regulates macrophage cell migration and polarization, as well as it is shown to modulate macrophage urokinase-type plasminogen activator (uPA) production, which also contributes to macrophage chemotaxis and migration toward damaged or inflamed tissues. Microtubule (MT) cytoskeleton dynamic plays a key role during the cell motility, and any interference on the MT network profoundly affects cell migration. In this study, by using estramustine phosphate (EP), which modifies MT stability, we analysed whether tubulin cytoskeleton contributes to TGF-β-induced macrophage cell migration and uPA expression. We found out that, in the murine macrophage cell line RAW 264.7, EP at noncytotoxic concentrations inhibited cell migration and uPA expression induced by TGF-β. Moreover, EP greatly reduced the capacity of TGF-β to trigger the phosphorylation and activation of its downstream Smad3 effector. Furthermore, Smad3 activation seems to be critical for the increased cell motility. Thus, our data suggest that EP, by interfering with MT dynamics, inhibits TGF-β-induced RAW 264.7 cell migration paralleled with reduction of uPA induction, in part by disabling Smad3 activation by TGF-β.

ER-localized protein-Herpud1 is a new mediator of IL-4-induced macrophage polarization and migration

ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response.

Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response.

Diabetic vasculopathy is intensified by macrophage inflammation caused by advanced glycation end products (AGEs). Heparanase (HPA) is a unique endoglycosidase, which cleaves heparan sulfate proteoglycans (HSPGs) including syndecan-1 (Syn-1) to further stimulate macrophage cell migration and inflammation. The present study was planned to evaluated the role of C-domain (if any) of HPA in AGE inflammatory response in macrophages. Cell viability was assessed using MTT assay, migration assay, ELISA for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) levels, mRNA expression by RT-PCR and heparan degrading enzyme assay for HPA activity. In the present study, we found that pretreatment with anti-HPA antibody, which recognizes the C-domain of HPA inhibited macrophage migration, secretion of IL-1β and TNF-α as well as decreased HPA enzymatic activity and increased Syn-1 protein expression in AGE-induced macrophages. Compared with anti-HPA antibody pretreatment, co-pretreatment with anti-HPA plus Syn-1 antibodies promoted macrophage migration, and secretion of IL-1β and TNF-α significantly in AGE-induced macrophages. In addition, pretreatment with anti-HPA or anti-HPA plus Syn-1 antibodies did not markedly change the mRNA levels of IL-1β and TNF-α concentration AGE-treated macrophages. The results showed that C-domain of HPA mediates AGE-induced macrophage migration and inflammatory cytokine release via Syn-1 protein expression. Furthermore, C-domain of HPA may have a key role in diabetic vascular complication-associated inflammatory response.

Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a.

Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of novel anti-inflammatory therapeutics.

657. Enhancing Therapeutic Efficacy of an Oncolytic Virus with a Beta-1 Integrin Blocking Antibody, OS2966

Glioblastoma Multiforme (GBM) is a destructive cancer of the central nervous system with poor outlook for its patients, which prompts for development of novel therapeutic approaches such as oncolytic viruses (OVs). OVs are genetically engineered viruses with tumor specific replication and lead to lysis and destruction of cancer cells. Our lab has developed a novel oncolytic Herpes Simplex Virus (oHSV), 34.5ENVE, which expresses the anti-angiogenic protein Vasculostatin. This virus has shown unparalleled antitumor efficacy against intracranial glioma in mice. OV therapy is limited by the activation and infiltration of monocytic cells that promote virus clearance and inhibit tumor destruction. β1 integrins are cell surface molecules involved in cellular proliferation, invasion and inflammation. We revealed that oHSV-induced cysteine-rich 61 protein (CCN1) binds with integrins on the surface of glioma cells and macrophages, triggering antiviral type-I interferon and chemokine responses that increase macrophage infiltration and activation, which causes increased virus clearance in glioblastoma cells and limits oncolytic virus efficacy. To improve OV therapeutic efficacy, we combined oHSV with OS2966, a β1 integrin neutralizing antibody, which has been humanized in anticipation of clinical trials. We hypothesized that OS2966 would mitigate CCN1- β1 integrin induced inflammation and increase oHSV replication. Combination treatment with OS2966 increased oHSV replication and glioma cell killing. Migration assays revealed that treatment with OS2966 strongly inhibited oHSV-induced raw246.7 macrophage cell migration toward infected glioblastoma cells. From our results, we demonstrate that β1 integrins limit oHSV replication. Furthermore, we concluded that OS2966 could rescue CCN1- β1 integrin mediated macrophage and microglia migration toward oHSV infected GBM cells. Our findings show how a β1 integrin neutralizing antibody (OS2966) inhibits the innate immune response triggered upon oHSV treatment. This work suggests that β1 integrin inhibition may be utilized in the development of a novel oHSV therapeutic strategy.

Abstract 361: Activation of Wnt Pathway in Macrophages During Atherosclerosis Regression

Introduction and Objective: Transcriptome analysis of plaque macrophages in two different mouse models of atherosclerosis regression revealed an over representation of consensus binding site sequences for the T-cell factor (TCF)/Lymphoid enhancer binding factor (LEF) family of transcription factors, suggesting canonical Wnt signaling pathway activation during regression in vivo. The canonical Wnt/β-catenin signaling pathway is important for cardiac development and regulates processes such as migration, invasion and tissue repair. However, its function in plaque macrophages is unclear. The objective of the study was to understand the role of canonical Wnt signaling in macrophages during regression using in vivo and in vitro approaches. Methods and Results: Immunohistochemistry of atherosclerotic arterial sections in mouse models of atherosclerosis regression (Reversa and aortic arch transplant) showed a significant increase in β-catenin expression in regressing vs. progressing macrophages. Elevated transcript levels of canonical Wnt downstream targets Ctnnb1, Lrp1 and Gja1 were detected in regressing plaque macrophages isolated by laser capture microdissection (LCM). Canonical Wnt signaling was further investigated in Wnt3a-stimulated primary bone marrow-derived macrophages (BMDM) in vitro, revealing upregulation of pathway target genes Ctnnb1 and Axin2. Furthermore, immunofluorescence analysis of BMDM stimulated with Wnt3a showed increased nuclear expression of β-catenin. Macrophage cell migration evaluated by scratch wound assay revealed a significant increase in migration in Wnt3a-treated vs. untreated BMDM. Conclusions: Our findings demonstrate that canonical Wnt signaling is activated in regressing plaque macrophages and regulates macrophage migration in vitro. Future studies are aimed at understanding the mechanism by which Wnt modulates macrophage migration.

WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in Drosophila.

Wiskott-Aldrich syndrome proteins (WASPs) are nucleation-promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution.

ET-70 * COMBINATION OF oHSV WITH INTEGRIN 1 NEUTRALIZING ANTIBODY (OS2966) AUGMENTS oHSV THERAPY

Oncolytic HSV (oHSV) is a biologic therapy currently being tested in patients. We have previously shown that oHSV-induced Cysteine-rich 61 protein (CCN1) binds with integrin α6β1, activating a type 1 IFN-antiviral defense response. oHSV-induced CCN1 induction also increases macrophage infiltration and activation, resulting in increased virus clearance in glioblastoma cells. Integrin β1 is cell surface molecule involved in multiple functions like cellular proliferation, invasion and inflammation. Inhibition of integrin β1 has shown anti-tumor efficacy in preclinical modles of glioma. Furthermore, incorporating the Rembrandt database of the National Cancer Institute, we found integrin β1 expression is negatively associated with glioblastoma patient survival (p value < 0.0001). Interestingly, oHSV infection strongly increased integrin β1 expression (western blot). Based on this, we hypothesized that the combination of oHSV with integrin β1neutralizing antibody (OS2966) will augment oHSV therapy. Combination treatment with OS2966 increased oHSV replication and cell killing. Increased viral replication and cell killing was more obvious in the laminin-coated cells, consistent with integrin β1 signaling pathway. OS2966 treatment strongly inhibited oHSV-induced raw246.7 macrophage cell migration towards infected glioblastoma cells as dose dependent manner (migration assay). Inhibition of integrin β1in glioma cell lines and patient derived primary GSCs significantly increased markers of sensencsence such as enhanced β-gal activity, along with induction of p21, and reduction of phosphorylation of AKT and Rb, indicating that integrin β1 activation is involved in self-renewal, and proliferation. In conclusion, oHSV-induced integrin β1 expression sensitize to OS2966 effect, resulting in enhanced viral replication and inhibition of macrophage migration. Our findings reveal how integrin β1 limits oHSV replication. Our findings also reveal how OS2966, an integrin β1 blocking antibody which has been humanized in anticipation of potential clinical trials, inhibits the innate immune response elicited upon oHSV therapy. The results will lead to the development of a novel oHSV therapeutic strategy.

Ontogenetic cell death and phagocytosis in the visual system of vertebrates.

Programmed cell death (PCD), together with cell proliferation, cell migration, and cell differentiation, is an essential process during development of the vertebrate nervous system. The visual system has been an excellent model on which to investigate the mechanisms involved in ontogenetic cell death. Several phases of PCD have been reported to occur during visual system ontogeny. During these phases, comparative analyses demonstrate that dying cells show similar but not identical spatiotemporally restricted patterns in different vertebrates. Additionally, the chronotopographical coincidence of PCD with the entry of specialized phagocytes in some regions of the developing vertebrate visual system suggests that factors released from degenerating cells are involved in the cell migration of macrophages and microglial cells. Contradicting this hypothesis however, in many cases the cell corpses generated during degeneration are rapidly phagocytosed by neighboring cells, such as neuroepithelial cells or Müller cells. In this review, we describe the occurrence and the sites of PCD during the morphogenesis and differentiation of the retina and optic pathways of different vertebrates, and discuss the possible relationship between PCD and phagocytes during ontogeny.

494: Prostaglandin E2 trans-activates the Colony-Stimulating Factor-1 (CSF-1) receptor and synergizes with CSF-1 in the induction of cell migration in macrophages via the mitogen-activated protein kinase ERK1/2

494: Prostaglandin E2 trans-activates the Colony-Stimulating Factor-1 (CSF-1) receptor and synergizes with CSF-1 in the induction of cell migration in macrophages via the mitogen-activated protein kinase ERK1/2

Abstract 329: Microparticles Induce Antiangiogenic Signaling in Microvascular Endothelial Cells by Activating a CD36-Dependent Signaling Pathway Involving Src Kinases and Reactive Oxygen Species

Cell-derived microparticles (MP), ≤1μm vesicles shed from vascular and blood cells during activation or apoptosis, circulate in plasma and are biomarkers of cardiovascular disease. The type 2 scavenger receptor CD36 binds MP via phosphatidylserine exposed on their surface. On microvascular endothelial cells (MVEC) CD36 induces anti-angiogenic signals via its cognate protein ligands, such as thrombospondin-1. We hypothesized that CD36-MP interactions on MVEC would inhibit angiogenesis. MP generated in vitro from THP1 cells dose dependently inhibited MVEC tube formation in in vitro matrigel angiogenesis assays (&gt;50% inhibition of number of tubes formed, branch points, and segments; p&lt;0.05). MP derived from human umbilical vein endothelial cells (HUVEC), MVEC and erythrocytes also inhibited tube formation showing that the anti-angiogenic effect of MP is independent of their cell of origin. THP1 MP also inhibited MVEC migration in a transwell assay by 80% (p&lt;0.001). Early passage MVEC with high CD36 expression were inhibited more than late passage MVEC with low CD36 expression (80% vs 33%; p&lt;0.001) establishing the specificity of CD36 in this process. Further, in HUVEC that do not express CD36, inhibition of migration by THP1 MP was only 44% (p&lt;0.001) but lentiviral-mediated heterologous expression of murine CD36 in HUVEC restored inhibition of migration to levels similar to that of MVEC (82%; p&lt;0.001), corroborating the specificity of CD36 in MP mediated inhibition of migration. Since CD36 dependent generation of reactive oxygen species (ROS) is necessary to inhibit cell migration in macrophages, we tested if MP can induce ROS in MVEC. THP1 MP dose dependently induced ROS in MVEC by two fold compared to control at 1hr (p&lt;0.05). NADPH oxidase inhibitor, apocynin, blocked MP induced ROS induction by &gt;95% and partially (33%) reversed the inhibition of tube formation in MVEC by THP1 MP (p&lt;0.05). Western blots of THP1 MP treated MVEC showed time-dependent activation of Fyn kinase, and PP2, a Src family kinase inhibitor, blocked MP induced ROS induction by &gt;95%. These studies identify a novel CD36 dependent mechanistic pathway through which cell-derived MP inhibit MVEC angiogenic activities through ROS generation involving NADPH oxidase and Src kinase Fyn.