Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics

The Adnectin CT-322 is a novel VEGF receptor 2 inhibitor that decreases tumor burden in an orthotopic mouse model of pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>Pancreatic cancer continues to have a 5-year survival of less than 5%. Therefore, more effective therapies are necessary to improve prognosis in this disease. Angiogenesis is required for tumor growth, and subsequently, mediators of angiogenesis are attractive targets for therapy. Vascular endothelial growth factor (VEGF) is a well-characterized mediator of tumor angiogenesis that functions primarily by binding and activating VEGF receptor 2 (VEGFR2). In this study, we evaluate the use of CT-322, a novel biologic (Adnectin). This small protein is based on a human fibronectin domain and has beneficial properties in that it is fully human, stable, and is produced in bacteria. CT-322 binds to and inhibits activation of VEGFR2.</p> <p>Methods</p> <p>The efficacy of CT-322 was evaluated <it>in vivo </it>using two orthotopic pancreatic tumor models. The first model was a human tumor xenograft where MiaPaCa-2 cells were injected into the tail of the pancreas of nude mice. The second model was a syngeneic tumor using Pan02 cells injected into pancreas of C57BL/6J mice. In both models, therapy was initiated once primary tumors were established. Mice bearing MiaPaCa-2 tumors were treated with vehicle or CT-322 alone. Gemcitabine alone or in combination with CT-322 was added to the treatment regimen of mice bearing Pan02 tumors. Therapy was given twice a week for six weeks, after which the animals were sacrificed and evaluated (grossly and histologically) for primary and metastatic tumor burden. Primary tumors were also evaluated by immunohistochemistry for the level of apoptosis (TUNEL), microvessel density (MECA-32), and VEGF-activated blood vessels (Gv39M).</p> <p>Results</p> <p>Treatment with CT-322 was effective at preventing pancreatic tumor growth and metastasis in orthotopic xenograft and syngeneic models of pancreatic cancer. Additionally, CT-322 treatment increased apoptosis, reduced microvessel density and reduced the number of VEGF-activated blood vessels in tumors. Finally, CT-322, in combination with gemcitabine was safe and effective at controlling the growth of syngeneic pancreatic tumors in immunocompetent mice.</p> <p>Conclusion</p> <p>We conclude that CT-322 is an effective anti-VEGFR2 agent and that further investigation of CT-322 for the treatment of pancreatic cancer is warranted.</p

r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction

Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors

GU81, a VEGFR2 antagonist peptoid, enhances the anti-tumor activity of doxorubicin in the murine MMTV-PyMT transgenic model of breast cancer

<p>Abstract</p> <p>Background</p> <p>Vascular endothelial growth factor (VEGF) is a primary stimulant of angiogenesis under physiological and pathological conditions. Anti-VEGF therapy is a clinically proven strategy for the treatment of a variety of cancers including colon, breast, lung, and renal cell carcinoma. Since VEGFR2 is the dominant angiogenic signaling receptor, it has become an important target in the development of novel anti-angiogenic therapies. We have reported previously the development of an antagonistic VEGFR2 peptoid (GU40C4) that has promising anti-angiogenic activity <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>In the current study, we utilize a derivative of GU40C4, termed GU81 in therapy studies. GU81 was tested alone or in combination with doxorubicin for <it>in vivo </it>efficacy in the MMTV-PyMT transgenic model of breast cancer.</p> <p>Results</p> <p>The derivative GU81 has increased <it>in vitro </it>efficacy compared to GU40C4. Single agent therapy (doxorubicin or GU81 alone) had no effect on tumor weight, histology, tumor fat content, or tumor growth index. However, GU81 is able to significantly to reduce total vascular area as a single agent. GU81 used in combination with doxorubicin significantly reduced tumor weight and growth index compared to all other treatment groups. Furthermore, treatment with combination therapy significantly arrested tumor progression at the premalignant stage, resulting in increased tumor fat content. Interestingly, treatment with GU81 alone increased tumor-VEGF levels and macrophage infiltration, an effect that was abrogated when used in combination with doxorubicin.</p> <p>Conclusion</p> <p>This study demonstrates the VEGFR2 antagonist peptoid, GU81, enhances the anti-tumor activity of doxorubicin in spontaneous murine MMTV-PyMT breast tumors.</p

Effects of Aging and Cyclosporin A on Collagen Turnover in Human Gingiva

BACKGROUND: WE AIMED AT CHARACTERIZING THE AGING GINGIVA ANALYZING: i) collagen content and turnover in human gingival tissues and fibroblasts obtained from healthy young and aging subjects. ii) the effect of cyclosporin A administration in human cultured gingival fibroblasts obtained from aging compared to young subjects. METHODS: Morphological analysis was performed on haematoxylin-eosin and Sirius red stained paraffin-embedded gingival biopsies from young and aging healthy subjects. The expression of the main genes and proteins involved in collagen turnover were determined by real time PCR, dot blot and SDS-zymography on cultured young and aging gingival fibroblasts, and after cyclosporin A administration. RESULTS: Our results suggest that in healthy aged people, gingival connective tissue is characterized by a similar collagen content and turnover. Collagen turnover pathways are similarly affected by cyclosporin A treatment in young and aging gingival fibroblasts. CONCLUSIONS: Cyclosporin A administration affects gingival collagen turnover pathways in young and aging fibroblasts at the same extent, suggesting that during aging cyclosporin A administration is not related to relevant collagen turnover modifications

SPARC promoter hypermethylation in colorectal cancers can be reversed by 5-Aza-2′deoxycytidine to increase SPARC expression and improve therapy response

Poor clinical outcomes in cancer can often be attributed to inadequate response to chemotherapy. Strategies to overcome either primary or acquired chemoresistance may ultimately impact on patients' survival favourably. We previously showed that lower levels of SPARC were associated with therapy-refractory colorectal cancers (CRC), and that upregulating its expression enhances chemo-sensitivity resulting in greater tumour regression in vivo. Here, we examined aberrant hypermethylation of the SPARC promoter as a potential mechanism for repressing SPARC in CRCs and whether restoration of its expression with a demethylating agent 5-Aza-2′deoxycytidine (5-Aza) could enhance chemosensitivity. Initially, the methylation status of the SPARC promoter from primary human CRCs were assessed following isolation of genomic DNA from laser capture microdissected specimens by direct DNA sequencing. MIP101, RKO, HCT 116, and HT-29 CRC cell lines were also used to evaluate the effect of 5-Aza on: SPARC promoter methylation, SPARC expression, the interaction between DNMT1 and the SPARC promoter (ChIP assay), cell viability, apoptosis, and cell proliferation. Our results revealed global hypermethylation of the SPARC promoter in CRCs, and identified specific CpG sites that were consistently methylated in CRCs but not in normal colon. We also demonstrate that SPARC repression in CRC cell lines could be reversed following exposure to 5-Aza, which resulted in increased SPARC expression, leading to a significant reduction in cell viability (by an additional 39% in RKO cells) and greater apoptosis (an additional 18% in RKO cells), when combined with 5-FU in vitro (in comparison to 5-FU alone). Our exciting findings suggest potential diagnostic markers of CRCs based on specific methylated CpG sites. Moreover, the results reveal the therapeutic utility of employing demethylating agents to improve response through augmentation of SPARC expression

HSPG-Binding Peptide Corresponding to the Exon 6a-Encoded Domain of VEGF Inhibits Tumor Growth by Blocking Angiogenesis in Murine Model

Vascular endothelial growth factor VEGF165 is a critical element for development of the vascular system in physiological and pathological angiogenesis. VEGF isoforms have different affinities for heparan sulphate proteoglycan (HSPG) as well as for VEGF receptors; HSPGs are important regulators in vascular development. Therefore, inhibition of interactions between VEGF and HSPGs may prevent angiogenesis. Here, we demonstrate that an HSPG-binding synthetic peptide, corresponding to exon 6a-encoded domain of VEGF gene, has anti-angiogenic property. This 20 amino acids synthetic peptide prevents VEGF165 binding to several different cell types, mouse embryonic sections and inhibits endothelial cell migration, despite its absence in VEGF165 sequence. Our in vivo anti-tumor studies show that the peptide inhibits tumor growth in both mouse Lewis-Lung Carcinoma and human Liposarcoma tumor-bearing animal models. This is the first evidence that a synthetic VEGF fragment corresponding to exon 6a has functional antagonism both in vitro and in vivo. We conclude that the above HPSG binding peptide (6a-P) is a potent inhibitor of angiogenesis-dependent diseases