Gene expression profile of the murine ischemic retina and its response to Aflibercept (VEGF-Trap)

Ischemic retinal dystrophies are leading causes of acquired vision loss. Although the dysregulated expression of the hypoxia-responsive VEGF-A is a major driver of ischemic retinopathies, implication of additional VEGF-family members in their pathogenesis has led to the development of multivalent anti-angiogenic tools. Designed as a decoy receptor for all ligands of VEGFR1 and VEGFR2, Aflibercept is a potent anti-angiogenic agent. Notwithstanding, the molecular mechanisms mediating Aflibercept’s efficacy remain only partially understood. Here, we used the oxygen-induced retinopathy (OIR) mouse as a model system of pathological retinal vascularization to investigate the transcriptional response of the murine retina to hypoxia and of the OIR retina to Aflibercept. While OIR severely impaired transcriptional changes normally ensuing during retinal development, analysis of gene expression patterns hinted at alterations in leukocyte recruitment during the recovery phase of the OIR protocol. Moreover, the levels of Angiopoietin-2, a major player in the progression of diabetic retinopathy, were elevated in OIR tissues and consistently downregulated by Aflibercept. Notably, GO term, KEGG pathway enrichment, and expression dynamics analyses revealed that, beyond regulating angiogenic processes, Aflibercept also modulated inflammation and supported synaptic transmission. Altogether, our findings delineate novel mechanisms potentially underlying Aflibercept’s efficacy against ischemic retinopathies.

Angiogenesis-Related Functions of Wnt Signaling in Colorectal Carcinogenesis

Aberrant activation of the Wnt/Fzd/β-catenin signaling pathway is one of the major molecular mechanisms of colorectal cancer (CRC) development and progression. On the other hand, one of the most common clinical CRC characteristics include high levels of angiogenesis, which is a key event in cancer cell dissemination and distant metastasis. The canonical Wnt/β-catenin downstream signaling regulates the most important pro-angiogenic molecules including vascular endothelial growth factor (VEGF) family members, matrix metalloproteinases (MMPs), and chemokines. Furthermore, mutations of the β-catenin gene associated with nuclear localization of the protein have been mainly detected in microsatellite unstable CRC. Elevated nuclear β-catenin increases the expression of many genes involved in tumor angiogenesis. Factors regulating angiogenesis with the participation of Wnt/β-catenin signaling include different groups of biologically active molecules including Wnt pathway components (e.g., Wnt2, DKK, BCL9 proteins), and non-Wnt pathway factors (e.g., chemoattractant cytokines, enzymatic proteins, and bioactive compounds of plants). Several lines of evidence argue for the use of angiogenesis inhibition in the treatment of CRC. In the context of this paper, components of the Wnt pathway are among the most promising targets for CRC therapy. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.

Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development

Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-IIhigh/trophic MHC-IIlow macrophage ratio was increased in tumors that produce anti-NRP-1 Nbs. This finding was corroborated by an increase in the expression of genes associated with MHC-IIhigh macrophages and a decrease in the expression of MHC-IIlow macrophage-associated genes in the macrophage pool sorted from anti-NRP-1 Nb-producing tumors. Moreover, we observed a significantly higher percentage of tumor-associated antigen-specific CD8+ T cells in tumors producing anti-NRP-1 Nbs. These data demonstrate that an intratumoral expression of NRP-1/Sema3A blocking biologicals increases anti-tumor immunity.

Role of VEGFs/VEGFR-1 Signaling and Its Inhibition in Modulating Tumor Invasion: Experimental Evidence in Different Metastatic Cancer Models

The vascular endothelial growth factor (VEGF) family members, VEGF-A, placenta growth factor (PlGF), and to a lesser extent VEGF-B, play an essential role in tumor-associated angiogenesis, tissue infiltration, and metastasis formation. Although VEGF-A can activate both VEGFR-1 and VEGFR-2 membrane receptors, PlGF and VEGF-B exclusively interact with VEGFR-1. Differently from VEGFR-2, which is involved both in physiological and pathological angiogenesis, in the adult VEGFR-1 is required only for pathological angiogenesis. Besides this role in tumor endothelium, ligand-mediated stimulation of VEGFR-1 expressed in tumor cells may directly induce cell chemotaxis and extracellular matrix invasion. Furthermore, VEGFR-1 activation in myeloid progenitors and tumor-associated macrophages favors cancer immune escape through the release of immunosuppressive cytokines. These properties have prompted a number of preclinical and clinical studies to analyze VEGFR-1 involvement in the metastatic process. The aim of the present review is to highlight the contribution of VEGFs/VEGFR-1 signaling in the progression of different tumor types and to provide an overview of the therapeutic approaches targeting VEGFR-1 currently under investigation.

Plasma levels of VEGF-A, VEGF B, and VEGFR-1 and applicability of these parameters as tumor markers in diagnosis of breast cancer

The VEGF family members are important factors in promoting angiogenesis and lymphangiogenesis in malignant processes. The aim of this study was to investigate plasma concentrations of VEGF-A, VEGF-B and their soluble VEGFR-1 receptor and their diagnostic utility and potency as compared to CA 15-3 in breast cancer patients and in relation to the control group. The study included 120 breast cancer patients and 60 control patients. Plasma levels of tested parameters were determined with ELISA and CA 15-3 levels were determined with CMIA. Concentrations of all tested parameters in breast cancer patients showed statistically significant difference when compared to the control groups (benign breast tumor patients and/or healthy women). VEGF-B showed the highest values of sensitivity (Sn) and predictive value of a negative test result (NPV) in total BC group (90% and 66.7%, respectively) and, more importantly, in stages I–II of BC (SE: 86.8%; 92.7%, NPV: 82.8%; 88.9%, respectively). Among all parameters tested, VEGF-A showed the highest specificity (Sf) (76.7%) and predictive value of a positive test result (PPV) (84.8%), yet they were lower than for CA 15-3. VEGF-A was also the best parameter that had statistically significant Area Under Curve (AUC) in stages I (0.678) and II (0.768). In the whole group of BC patients all parameters tested showed statistically significant AUC, but the maximum range was obtained for the combination of VEGF-A and CA 15-3 (0.817). The combined analysis of the studied parameters and CA 15-3 resulted in an increase in sensitivity and AUC values, which provides hope for developing a new panel of biomarkers that may be used in BC diagnosis in the future.

VEGF-B is a potent antioxidant

VEGF-B was discovered a long time ago. However, unlike VEGF-A, whose function has been extensively studied, the function of VEGF-B and the mechanisms involved still remain poorly understood. Notwithstanding, drugs that inhibit VEGF-B and other VEGF family members have been used to treat patients with neovascular diseases. It is therefore critical to have a better understanding of VEGF-B function and the underlying mechanisms. Here, using comprehensive methods and models, we have identified VEGF-B as a potent antioxidant. Loss of Vegf-b by gene deletion leads to retinal degeneration in mice, and treatment with VEGF-B rescues retinal cells from death in a retinitis pigmentosa model. Mechanistically, we demonstrate that VEGF-B up-regulates numerous key antioxidative genes, particularly, Gpx1. Loss of Gpx1 activity largely diminished the antioxidative effect of VEGF-B, demonstrating that Gpx1 is at least one of the critical downstream effectors of VEGF-B. In addition, we found that the antioxidant function of VEGF-B is mediated mainly by VEGFR1. Given that oxidative stress is a crucial factor in numerous human diseases, VEGF-B may have therapeutic value for the treatment of such diseases.

Correlation of tissue plasminogen regulators expressed by esophageal adenocarcinoma with VEGF family members.

37 Background: Esophageal cancer is known for its high potential of early lymphogenous metastases. The biological activity of plasminogen (PG) regulators, VEGFs and their receptors controls the growth and differentiation of cells, including malignant ones, the stability of the extracellular matrix, as well as processes of destruction of membranes and extracellular matrix, invasion of malignant cells, angio- and lymphangiogenesis. Our purpose was to study the role of PG regulators in the activation of VEGFs and their receptors in esophageal adenocarcinoma (EA) and its perifocal zone (PZ). Methods: Levels of uPA-Ag, uPA-act; tPA-Ag, tPA-act; PAI-1-Ag and PAI-1-act, VEGF-А, VEGF-R1, VEGF-С and VEGF-R3 were determined by ELISA in surgical specimens of EA (n = 28, st II, G2, T2-3N0-1M0). Statistical analysis was performed using Microsoft Office Excel 2010. Results: Levels of all proteins, except tPA, were higher in tumor (T) tissues than in the resection line (RL, p < 0.01). Protein levels, except uPA-Ag and tPA, in PZ were between levels in T and RL (p < 0.01); uPA-Ag, tPA-Ag and tPA-act were decreased in T and PZ compared with RL (p < 0.01); PAI-1-Ag in T and PZ was higher than in RL by 9.4 and 6.3 times, and PAI-1-act – by 10.7 and 1.9 times. Levels of VEGF-А, VEGF-R1, VEGF-С and VEGF-R3 were increased in T (p < 0.001), and in PZ they were between levels in T and RL (p < 0.001). Strong correlations were registered in EA T between uPA and VEGF-А (r = 86), uPA and VEGF-R1 (r = 81), uPA and VEGF-С (r = 79), uPA and VEGF-R3 (r = 76). A strong correlation between uPA and PAI-1 in T (r = 88) did not exclude PAI-1 biological effects in EA T and its PZ. PZ showed strong correlations between uPA and PAI-1 (r = 87), uPA and VEGF-А (r = 84), uPA and VEGF-С (r = 78). Conclusions: The strong correlations between the activation of uPA, PAI-1, VEGF-А, VEGF-С and their receptors in EA indicate interactions between them, realized both directly and through plasmin. The expression of the studied proteins in T and its PZ, compared to RL, demonstrates the effect of T on the content and activity of uPA, PAI-1, VEGF-A, VEGF-C and their receptors, with the following realization of biological properties that promote the progression of EA.

The lymphatic system

The lymphatic vascular system is an integral component of the circulatory system; it forms a one-way conduit that transports tissue interstitial components back to the venous circulation through lymph nodes. Lymphatic vessels extend to most tissues and contribute to the regulation of interstitial fluid homeostasis, trafficking of immune cells, and absorption of dietary fats from the gut. Developmentally, lymphatic vessels originate from embryonic veins and specialized angioblasts. A number of molecules have been identified in the commitment of endothelial cells to the lymphatic lineage, and the sprouting, expansion and maturation of the lymphatic vascular tree. Importantly, the vascular endothelial growth factor (VEGF) family members VEGFC and VEGFD, together with their receptors VEGFR2 and VEGFR3 have been implicated as critical regulators of lymphangiogenesis. Lymphatic vessels are involved in several human diseases, including cancer, where they contribute to tumour metastasis, the leading cause of cancer-related deaths. Lymphatic vessels regulate immune responses against foreign pathogens by transporting leucocytes to lymph nodes, but are also in involved in the regulation of self-tolerance. Defects in the lymphatic vascular system are causal for the development of lymphoedema.

Regulation of Vegf signaling by natural and synthetic ligands

The mechanisms that allow cells to bypass anti–vascular endothelial growth factor A (VEGFA) therapy remain poorly understood. Here we use zebrafish to investigate this question and first show that vegfaa mutants display a severe vascular phenotype that can surprisingly be rescued to viability by vegfaa messenger RNA injections at the 1-cell stage. Using vegfaa mutants as an in vivo test tube, we found that zebrafish Vegfbb, Vegfd, and Pgfb can also rescue these animals to viability. Taking advantage of a new vegfr1 tyrosine kinase–deficient mutant, we determined that Pgfb rescues vegfaa mutants via Vegfr1. Altogether, these data reveal potential resistance routes against current anti-VEGFA therapies. In order to circumvent this resistance, we engineered and validated new dominant negative Vegfa molecules that by trapping Vegf family members can block vascular development. Thus, our results show that Vegfbb, Vegfd, and Pgfb can sustain vascular development in the absence of VegfA, and our newly engineered Vegf molecules expand the toolbox for basic research and antiangiogenic therapy.

Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.