Olfactomedin-like 3 promotes PDGF-dependent pericyte proliferation and migration during embryonic blood vessel formation.

Pericytes promote vessel stability and their dysfunction causes pathologies due to blood vessel leakage. Previously, we reported that Olfactomedin-like 3 (Olfml3) is a matricellular protein with proangiogenic properties. Here, we explored the role of Olfml3 in a knockout mouse model engineered to suppress this protein. The mutant mice exhibited vascular defects in pericyte coverage, suggesting that pericytes influence blood vessel formation in an Olfml3-dependent manner. Olfml3-deficient mice exhibited abnormalities in the vasculature causing partial lethality of embryos and neonates. Reduced pericyte coverage was observed at embryonic day 12.5 and persisted throughout development, resulting in perinatal death of 35% of Olfml3-deficient mice. Cultured Olfml3-deficient pericytes exhibited aberrant motility and altered pericyte association to endothelial cells. Furthermore, the proliferative response of Olfml3-/- pericytes upon PDGF-B stimulation was significantly diminished. Subsequent experiments revealed that intact PDGF-B signaling, mediated via Olfml3 binding, is required for pericyte proliferation and activation of downstream kinase pathways. Our findings suggest a model wherein pericyte recruitment to endothelial cells requires Olfml3 to provide early instructive cue and retain PDGF-B along newly formed vessels to achieve optimal angiogenesis.

Junctional adhesion molecule B interferes with angiogenic VEGF/VEGFR2 signaling.

De novo formation of blood vessels is a pivotal mechanism during cancer development. During the past few years, antiangiogenic drugs have been developed to target tumor vasculature. However, because of limitations and adverse effects observed with current therapies, there is a strong need for alternative antiangiogenic strategies. Using specific anti-junctional adhesion molecule (JAM)-B antibodies and Jam-b-deficient mice, we studied the role in antiangiogenesis of JAM-B. We found that antibodies against murine JAM-B, an endothelium-specific adhesion molecule, inhibited microvessel outgrowth from ex vivo aortic rings and in vitro endothelial network formation. In addition, anti-JAM-B antibodies blocked VEGF signaling, an essential pathway for angiogenesis. Moreover, increased aortic ring branching was observed in aortas isolated from Jam-b-deficient animals, suggesting that JAM-B negatively regulates proangiogenic pathways. In mice, JAM-B expression was detected in de novo-formed blood vessels of tumors, but anti-JAM-B antibodies unexpectedly did not reduce tumor growth. Accordingly, JAM-B deficiency in vivo had no impact on blood vessel formation, suggesting that targeting JAM-B in vivo may be offset by other proangiogenic mechanisms. In conclusion, despite the promising effects observed in vitro, targeting JAM-B during tumor progression seems to be inefficient as a stand-alone antiangiogenesis therapy.

Biodegradable and plasma-treated electrospun scaffolds coated with recombinant Olfactomedin-like 3 for accelerating wound healing and tissue regeneration.

Three-dimensional biomimetic scaffolds resembling the native extracellular matrix (ECM) are widely used in tissue engineering, however they often lack optimal bioactive cues needed for acceleration of cell proliferation, neovascularization, and tissue regeneration. In this study, the use of the ECM-related protein Olfactomedin-like 3 (Olfml3) demonstrates the importance and feasibility of fabricating efficient bioactive scaffolds without in vitro cell seeding prior to in vivo implantation. First, in vivo proangiogenic properties of Olfml3 were shown in a murine wound healing model by accelerated wound closure and a 1.4-fold increase in wound vascularity. Second, subcutaneous implantation of tubular scaffolds coated with recombinant Olfml3 resulted in enhanced cell in-growth and neovascularization compared with control scaffolds. Together, our data indicates the potential of Olfml3 to accelerate neovascularization during tissue regeneration by promoting endothelial cell proliferation and migration. This study provides a promising concept for the reconstruction of damaged tissue using affordable and effective bioactive scaffolds.

PTPRZ1-targeting RNA CAR T cells exert antigen-specific and bystander antitumor activity in glioblastoma.

The great success of chimeric antigen receptor (CAR) T-cell therapy in the treatment of patients with B-cell malignancies has prompted its translation to solid tumors. In the case of glioblastoma (GBM), clinical trials have shown modest efficacy, but efforts to develop more effective anti-GBM CAR T cells are ongoing. In this study, we selected PTPRZ1 as a target for GBM treatment. We isolated six anti-human PTPRZ1 scFv from a human phage display library and produced 2nd generation CAR T cells in an RNA format. Patient-derived GBM PTPRZ1-knock-in cell lines were used to select the CAR construct that showed high cytotoxicity while consistently displaying high CAR expression (471_28z). CAR T cells incorporating 471_28z were able to release IFN-γ, IL-2, TNF-α, Granzyme B, IL-17A, IL-6, and soluble FasL, and displayed low tonic signaling. Additionally, they maintained an effector memory phenotype after in vitro killing. In addition, 471_28z CAR T cells displayed strong bystander killing against PTPRZ1-negative cell lines after pre-activation by PTPRZ1-positive tumor cells but did not kill antigen-negative non-tumor cells. In an orthotopic xenograft tumor model using NSG mice, a single dose of anti-PTPRZ1 CAR T cells significantly delayed tumor growth. Taken together, these results validate PTPRZ1 as a GBM target and prompt the clinical translation of anti-PTPRZ1 CAR T cells.

A quantitative comparison of antibodies against epitope tags for immunofluorescence detection.

Epitope tags recognized by specific antibodies have been widely used over the last few decades, notably to localize tagged proteins within cells by immunofluorescence. The diversity of tags and antibodies usually prevents a side-by-side comparison of the efficiency with which each antibody recognizes its cognate tag. We expressed chimeric proteins, each composed of an invariant domain (IL2Ra) associated with a specific epitope tag. Double immunofluorescence allowed us to quantify in parallel the reference signal generated by the anti-IL2Ra antibody and the signal generated by the anti-epitope tag antibody. Since all antibodies used in this study were recombinant antibodies fused to the same mouse Fc domain, the generated signals were directly comparable. Three groups of tags/antibodies were revealed: 'good' antibodies generated high signals even when used at a low concentration (50 ng·mL-1 ), 'fair' antibodies generated a high signal only at high concentrations (5000 ng·mL-1 ), and 'mediocre' antibodies generated positive but weak signals. Except for an anti-myc antibody, similar results were obtained when cells were fixed in paraformaldehyde or methanol. These results provide a side-by-side quantitative evaluation of different tag/antibody pairs. This information will be useful to optimize the choice of epitope tags and to choose optimal antibodies.

Intracellular targeting of Cisd2/Miner1 to the endoplasmic reticulum.

BACKGROUND: Cisd1 and Cisd2 proteins share very similar structures with an N-terminal membrane-anchoring domain and a C-terminal cytosolic domain containing an iron-cluster binding domain and ending with a C-terminal KKxx sequence. Despite sharing a similar structure, Cisd1 and Cisd2 are anchored to different compartments: mitochondria for Cisd1 and endoplasmic reticulum for Cisd2. The aim of this study was to identify the protein motifs targeting Cisd2 to the ER and ensuring its retention in this compartment. RESULTS: We used new recombinant antibodies to localize Cisd1 and Cisd2 proteins, as well as various protein chimeras. Cisd2 is targeted to the ER by its N-terminal sequence. It is then retained in the ER by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif, and of an ER-targeting transmembrane domain. As previously reported for Cisd1, Cisd2 can alter the morphology of the compartment in which it accumulates. CONCLUSION: Although they share a very similar structure, Cisd1 and Cisd2 use largely different intracellular targeting motifs to reach their target compartment (mitochondria and endoplasmic reticulum, respectively).

Targeting OLFML3 in Colorectal Cancer Suppresses Tumor Growth and Angiogenesis, and Increases the Efficacy of Anti-PD1 Based Immunotherapy.

The role of the proangiogenic factor olfactomedin-like 3 (OLFML3) in cancer is unclear. To characterize OLFML3 expression in human cancer and its role during tumor development, we undertook tissue expression studies, gene expression analyses of patient tumor samples, in vivo studies in mouse cancer models, and in vitro coculture experiments. OLFML3 was expressed at high levels, mainly in blood vessels, in multiple human cancers. We focused on colorectal cancer (CRC), as elevated expression of OLFML3 mRNA correlated with shorter relapse-free survival, higher tumor grade, and angiogenic microsatellite stable consensus molecular subtype 4 (CMS4). Treatment of multiple in vivo tumor models with OLFML3-blocking antibodies and deletion of the Olfml3 gene from mice decreased lymphangiogenesis, pericyte coverage, and tumor growth. Antibody-mediated blockade of OLFML3 and deletion of host Olfml3 decreased the recruitment of tumor-promoting tumor-associated macrophages and increased infiltration of the tumor microenvironment by NKT cells. Importantly, targeting OLFML3 increased the antitumor efficacy of anti-PD-1 checkpoint inhibitor therapy. Taken together, the results demonstrate that OLFML3 is a promising candidate therapeutic target for CRC.

Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of glioma.

Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas.

Sushi repeat protein X-linked 2, a novel mediator of angiogenesis.

On appropriate stimuli, quiescent endothelial cells start to proliferate and form de novo blood vessels through angiogenesis. To further define molecular mechanisms accompanying the activation of endothelial cells during angiogenesis, we identified genes that were differentially regulated during this process using microarray analyses. In this work, we established a regulatory role for Sushi repeat protein X-linked 2 (Srpx2) in endothelial cell remodeling during angiogenesis. In particular, silencing of Srpx2 using small interfering RNAs (siRNAs) specifically attenuated endothelial cell migration and delayed angiogenic sprout formation. In vivo, Srpx2 expression was detected in de novo formation of blood vessels in angiogenic tissues by in situ mRNA hybridization and immunostaining. Pulldown experiments identified Srpx2 as a ligand for vascular uPAR, a key molecule involved in invasive migration of angiogenic endothelium. Immunostaining revealed coexpression of the Srpx2 and uPAR on vascular endothelium. These findings suggest that Srpx2 regulates endothelial cell migration and tube formation and provides a new target for modulating angiogenesis.

A recombinant antibody toolbox for Dictyostelium discoideum.

OBJECTIVE: The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion, macropinocytosis and phagocytosis, host-pathogen interactions and multicellular development. However, the relative small size of the Dictyostelium community hampers the production and distribution of reagents and tools, such as antibodies, by commercial vendors. RESULTS: For the past 5 years, our laboratory has worked to promote an increased use of recombinant antibodies (rAbs) by academic laboratories. Here we report our efforts to ensure that Dictyostelium researchers have access to rAbs. Using hybridoma sequencing and phage display techniques, we generated a panel of recombinant antibodies against D. discoideum antigens, providing a useful and reliable set of reagents for labelling and characterization of proteins and subcellular compartments in D. discoideum, accessible to the entire Dictyostelium community.

Generation of Monoclonal Antibodies Specific for Native LL37 and Citrullinated LL37 That Discriminate the Two LL37 Forms in the Skin and Circulation of Cutaneous/Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients.

Human cathelicidin LL37 is a cationic antimicrobial peptide active against bacteria and viruses and exerting immune modulatory functions. LL37 can be also a target of autoreactive B- and T-lymphocytes in autoimmune settings. Irreversible post-translational modifications, such as citrullination and carbamylation, mainly occurring at the level of cationic amino acids arginine and lysine, can affect the inflammatory properties and reduce antibacterial effects. Moreover, these modifications could be implicated in the rupture of immune tolerance to LL37 in chronic conditions such as psoriatic disease and cutaneous lupus (LE)/systemic lupus erythematosus (SLE). Here, we describe the generation and fine specificity of six recombinant antibodies (MRB137-MRB142), produced as a monovalent mouse antibody with the antigen-binding scFv portion fused to a mouse IgG2a Fc, and their ability to recognize either native or citrullinated LL37 (cit-LL37) and not cross-react to carbamylated LL37. By using these antibodies, we detected native LL37 or cit-LL37 in SLE and rheumatoid arthritis (RA) sera, and in LE skin, by ELISA and immunohistochemistry, respectively. Such antibodies represent previously unavailable and useful tools to address relationships between the presence of post-translational modified LL37 and the immune system status (in terms of innate/adaptive responses activation) and the clinical characteristics of patients affected by chronic immune-mediated diseases or infectious diseases.

Targeting olfactomedin-like 3 inhibits tumor growth by impairing angiogenesis and pericyte coverage.

Antiangiogenic drugs have been used as anticancer agents to target tumor endothelial cells or pericytes. Because of limited efficacy of the current monotherapies, there is a strong demand for the dual targeting of endothelial cells and pericytes. Here, we identify Olfactomedin-like 3 (Olfml3) as a novel proangiogenic cue within the tumor microenvironment. Tumor-derived Olfml3 is produced by both tumor endothelial cells and accompanying pericytes and deposited in the perivascular compartment. Blockade of Olfml3 by anti-Olfml3 antibodies is highly effective in reducing tumor vascularization, pericyte coverage, and tumor growth. In vitro, Olfml3 targeting is sufficient to inhibit endothelioma cell migration and sprouting. Olfml3 alone or through binding to BMP4 enhances the canonical SMAD1/5/8 signaling pathway required for BMP4-induced angiogenesis. Therefore, Olfml3 blockade provides a novel strategy to control tumor growth by targeting two distinct cell types within the tumor microenvironment using a single molecule.

Expression and function of junctional adhesion molecule-C in human and experimental arthritis.

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule involved in transendothelial migration of leukocytes. In this study, we examined JAM-C expression in the synovium and investigated the role of this molecule in two experimental mouse models of arthritis. JAM-C expression was investigated by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of a monoclonal anti-JAM-C antibody were assessed in antigen-induced arthritis (AIA) and K/BxN serum transfer-induced arthritis. JAM-C was expressed by synovial fibroblasts in the lining layer and associated with vessels in the sublining layer in human and mouse arthritic synovial tissue. In human tissue, JAM-C expression was increased in rheumatoid arthritis (RA) as compared to osteoarthritis synovial samples (12.7 +/- 1.3 arbitrary units in RA versus 3.3 +/- 1.1 in OA; p < 0.05). Treatment of mice with a monoclonal anti-JAM-C antibody decreased the severity of AIA. Neutrophil infiltration into inflamed joints was selectively reduced as compared to T-lymphocyte and macrophage infiltration (0.8 +/- 0.3 arbitrary units in anti-JAM-C-treated versus 2.3 +/- 0.6 in isotype-matched control antibody-treated mice; p < 0.05). Circulating levels of the acute-phase protein serum amyloid A as well as antigen-specific and concanavalin A-induced spleen T-cell responses were significantly decreased in anti-JAM-C antibody-treated mice. In the serum transfer-induced arthritis model, treatment with the anti-JAM-C antibody delayed the onset of arthritis. JAM-C is highly expressed by synovial fibroblasts in RA. Treatment of mice with an anti-JAM-C antibody significantly reduced the severity of AIA and delayed the onset of serum transfer-induced arthritis, suggesting a role for JAM-C in the pathogenesis of arthritis.