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Macrophages play important roles in angiogenesis; however, previous studies on macrophage angiogenesis have focused on traditional 2D cultures. In this study, we established a 3D culture system for macrophages using collagen microcarriers and assessed the effect of 3D culture on their angiogenic capabilities. Macrophages grown in 3D culture displayed a significantly different morphology and arrangement under electron microscopy compared to those grown in 2D culture. Tube formation assays and chick embryo chorioallantoic membrane assays further revealed that 3D-cultured macrophages were less angiogenic than those in 2D culture. Whole-transcriptome sequencing showed that nearly 40% of genes were significantly differently expressed, including nine important angiogenic factors of which seven had been downregulated. In addition, the expression of almost all genes related to two important angiogenic pathways was decreased in 3D-cultured macrophages, including the two key angiogenic factors, VEGFA and ANG2. Together, the findings of our study improve our understanding of angiogenesis and 3D macrophage culture in tissues, and provide new avenues and methods for future research on macrophages.

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects1–4. To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examinedin vivo, superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.

The guarantee of cell survival under hypoxic conditions and rapid vascularization is a key in tissue engineering strategies for treating bone defects. Our study aimed to establish the protective role of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) in hypoxic conditions and realize rapid vascularization in bone defects. Resveratrol (Res), a non-flavonoid polyphenolic compound, and angiopoietin-2 (ANG2), a vascular activating factor, were applied to enhance BMSC and HUVEC survival, osteogenesis, and angiogenesis. The morphology, autophagy, viability, apoptosis, cycle, and osteogenic differentiation of BMSCs treated with Res were analyzed. The results indicated that Res could improve BMSC survival and differentiation via the autophagy pathway under hypoxic conditions. In addition, Res maintained HUVEC growth and proliferation in a hypoxic and ANG2 double-adverse environment via the autophagy pathway. To simulate a relatively hypoxic environment, small-aperture PEGDA/TCS hydrogels containing Res and ANG2 were prepared. BMSCs were cultured in the PEGDA/TCS scaffold and transplanted into a large tibial defect. CD31 immunofluorescence showed that the density and size of new blood vessels in the bone defect were significantly enhanced by ANG2 and Res at 8 weeks after surgery. H&E, Masson, and immunohistochemical staining results indicated that ANG2 combined with Res could promote new bone formation in defects. All these results suggested that Res combined with ANG2 may be a novel strategy for the targeted therapy of hypoxic bone defects with tissue engineering scaffolds.

The rhythm gene BMAL1 (Brain and Muscle ARNT-Like 1) may play an important role in glioma tolerance for anti-angiogenesis therapy. In humans with glioma of different pathological grades, BMAL1 expression was significantly different, and the expression of ANG2 (Angiopoietin 2) and VEGF (Vascular endothelial growth factor) was positively correlated with the expression of BMAL1. Additionally, BMAL1 expression is positively correlated with the microvascular density and peritumoral edema of glioma. According to in vitro experiments, silencing the expression of BMAL1 in primary glioma cells results in a decrease in the expression of VEGF. In contrast, overexpression of BMAL1 promotes the expression of ANG2 and VEGF via HIF-1a pathway. Therefore, BMAL1 likely participates in the angiogenesis of glioma by modulating ANG2 and VEGF expression, alters the therapeutic effect of anti-angiogenic treatments, and promotes peritumoral brain edema of glioma.

Intravitreal antivascular endothelial growth factor (VEGF) drugs represent the first-line treatment option for wet age-related macular degeneration (w-AMD) and diabetic macular edema (DME); however, the frequent injection intervals have illuminated to the necessity for new molecules allowing a more prolonged treatment regimen. Faricimab is a promising bispecific drug targeting VEGF-A and the Ang-Tie/pathway. Phase II STAIRWAY and AVENUE Trials showed its clinical efficacy for the treatment of w-AMD, while the phase II BOULEVARD Trial revealed its superiority to monthly ranibizumab in the management of DME with a monthly treatment regimen. The agents are awaiting approval for the treatment of w-AMD and DME.This article presents an overview of w-AMD and diabetic retinopathy and examines the progress of Faricimab through clinical trials. It offers insights on where Faricimab may be placed in the future market of anti-VEGF treatments and discusses the role of Ang/Tie pathway as a potential additive weapon for the treatment of w-AMD, DME, and retinal vein occlusion (RVO).The possibility of administering faricimab with more prolonged treatment intervals represents an important advantage to decrease the treatment burden and improve patient compliance. Further phase III trials should provide more evidence on clinical efficacy.

Background Proper oxygen balance in the dental pulp is essential for cell metabolism. Angiogenesis in the pulp is a constant process during the life of the tooth. Hypoxia indicators in a tissue, such as HIF-1α, as well as vascular destabilization markers, such as ANG2 and its receptor TIE2, are necessary for angiogenesis. Therefore the purpose of this study is to evaluate the expression of HIF-1α, ANG1, ANG2 and TIE2 in dental pulp as early angiogenesis indicators in teeth with complete and incomplete root development. Methods Forty human dental pulps were obtained from freshly extracted third molars divided into two groups: incomplete (n = 20) and complete (n = 20) root development. Dental pulps were stored at − 80 °C, defrosted in an ice bath and re-frozen with their respective thaws to disintegrate the tissue. Three sonication cycles were performed until the tissues were homogenized, then thaw were centrifuged and the supernatant was collected for the detection of the markers to be studied. The samples were processed for the ELISA test using the ELISA-sandwich principle. Student t and Mann–Whitney U tests were performed to determine statistically significant differences between groups. Results In the complete root development, HIF-1α, ANG1, ANG2 and TIE2 expressions were significantly higher than their expression in the incomplete root development group. Conclusions The angiogenic process seems to be a physiological process in the dental pulp. Angiogenic activity is higher in teeth with mature than immature apex teeth.

Background Cavity effusion is common in patients with infectious diseases. However, the incidence rate and characteristics of serous cavity effusions (SCE) in septic patients are not clear to date. The objective of this study was to investigate the incidence and characteristics of SCE in septic patients and to explore the correlations between the bloody effusions and the illness severity/prognosis in septic patients. Methods From January 2010 to January 2015, a total of 214 patients with severe sepsis and septic shock were enrolled in this retrospective observational study. Thoracentesis or abdominal paracentesis was performed in 45 septic patients because of massive pleural effusions or ascites. The serum concentrations of VEGF, VEGFR, Ang, sICAM-1, sVCAM-1, E-selectin, Serpine1 and VE-cadherin in 45 septic patients underwent paracentesis were measured by enzyme-linked immunosorbent assay (ELISA). Results Of the 214 septic patients, 155 (72.4%) had SCE according to imaging or ultrasound manifestations. 45 subjects with SCE underwent therapeutic thoracentesis or abdominal paracentesis. Effusion laboratory analysis showed that exudates were predominant when compared with transudates (95.6% vs. 4.4%), and 16 (35.6%) patients suffered bloody effusions. Compared with patients with non-bloody effusions, those with bloody effusions showed higher critical illness scores (13 vs. 17 for APACHE II; 7 vs. 9 for SOFA), and higher mortality (6.9% vs. 62.5%). Moreover, patients with bloody effusions had delayed TT and APTT, increased D-dimer concentration, and higher serum levels of CRP and PCT (P

Background Choroidal neovascularization (CNV) is a major cause of blindness in patients with age-related macular degeneration. CNV is characterized by new blood vessel growth and subretinal fluid accumulation, which results in mechanical pressure on retinal pigment epithelial (RPE) cells. The overexpression of RPE-derived angiogenic factors plays an important role in inducing CNV. In this work, we investigated the effect of mechanical stress on the expression of angiogenic factors in porcine RPE cells and determined the impact of conditioned medium on in-vitro angiogenesis. Results The goal of this study was to determine whether low levels of acute mechanical stress during early CNV can induce the expression of angiogenic factors in RPE cells and accelerate angiogenesis. Using a novel device, acute mechanical stress was applied to primary porcine RPE cells and the resulting changes in the expression of major angiogenic factors, VEGF, ANG2, HIF-1α, IL6, IL8 and TNF-α, were examined using immunocytochemistry, qRT-PCR, and ELISA. An in vitro tube formation assay was used to determine the effect of secreted angiogenic proteins due to mechanical stress on endothelial tube formation by human umbilical vein endothelial cells (HUVECs). Our results showed an increase in the expression of VEGF, ANG2, IL-6 and IL-8 in response to mechanical stress, resulting in increased in vitro angiogenesis. Abnormal epithelial-mesenchymal transition (EMT) in RPE cells is also associated with CNV and further retinal degeneration. Our qRT-PCR results verified an increase in the expression of EMT genes, CDH2, VIM and FN1, in RPE cells. Conclusions In conclusion, we showed that acute mechanical stress induces the expression of major angiogenic and EMT factors and promotes in vitro angiogenesis, suggesting that mechanical stress plays a role in promoting aberrant angiogenesis in AMD.

Sepsis is associated with significant mortality. Early diagnosis and prognosis of patients with sepsis is still a difficult clinical challenge. In this study, the ability of plasma PTX3 (pentraxin 3), MCP1 (monocyte chemoattractant protein 1) and Ang (angiopoietin)1/2 was investigated to evaluate the severity of sepsis. Blood samples were obtained from 43 patients with sepsis. A total of 33 post‐surgery patients with infections and 25 healthy individuals served as controls. The results showed that plasma PTX3, MCP1 and Ang2 significantly increased in patients on the first day of septic shock onset, while sepsis patients had significantly higher Ang2 level, compared with controls. Furthermore, PTX3, MCP1 and Ang2 had high AUROC values in patients with septic shock on the first day of sepsis onset. The findings suggest that PTX3, MCP1 and Ang2 maybe early predictors to evaluate the severity of sepsis and septic shock with the latest Sepsis 3.0 definitions.

Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)–Tie system is a second endothelial cell specific ligand–receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang–Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang–Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang–Tie system in vascular development and pathogenesis of vascular diseases.

Background/Aims: The pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins (Angs) play a prominent role in synovial angiogenesis, an early and critical event in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-35 is an anti-inflammatory cytokine that attenuates collagen-induced arthritis, however, the mechanisms involved are not fully understood. Methods: The effects of IL-35 on endothelial cell migration, adhesion, and tube formation were examined using human umbilical vein endothelial cells (HUVEC) in vitro. The effects of IL-35 on vessel formation in vivo were examined using a murine Matrigel plugs model. MMP2/MMP9 and IL-6/IL-8 secretion were assessed by zymography and ELISA, respectively. The crosstalk between IL-35, VEGF, and Ang2 in HUVECs and RA synovial tissue explants was investigated. Results: IL-35 inhibited basal and VEGF-induced HUVEC migration and adhesion in vitro as well as tube formation in vitro and in vivo. VEGF increased Ang2 secretion by HUVECs and RA synovial tissue explants, and exogenous Ang2 promoted HUVEC migration, adhesion, and tube formation with similar potency to VEGF. Blocking the Ang/Tie2 pathway with a Tie2 kinase antibody inhibited the proangiogenic effects of exogenous Ang2 and VEGF in HUVECs. IL-35 inhibited basal and VEGF-induced Ang2 secretion by HUVECs and RA synovial tissue explants; it also antagonized the proangiogenic effects of exogenous Ang2 in HUVECs. Moreover, IL-35 reduced basal and VEGF/Ang2-induced MMP2/MMP9 and IL-6/IL-8 secretion. Conclusion: These results suggested that IL-35 restrains RA angiogenesis and inflammation by downregulating basal and VEGF-induced Ang2 secretion as well as disrupting Ang2/Tie2 signal transduction. Our findings extend current understanding of mechanisms regulating RA angiogenesis and may support development of novel angiogenesis-targeting therapeutics for RA treatment.

Despite significant advances made in cancer treatment, the development of therapeutic resistance to anticancer drugs represents a major clinical problem that limits treatment efficacy for cancer patients. Herein, we focus on the response and resistance to current antiangiogenic drugs and immunotherapies and describe potential strategies for improved treatment outcomes. Antiangiogenic treatments that mainly target vascular endothelial growth factor (VEGF) signaling have shown efficacy in many types of cancer. However, drug resistance, characterized by disease recurrence, has limited therapeutic success and thus increased our urgency to better understand the mechanism of resistance to inhibitors of VEGF signaling. Moreover, cancer immunotherapies including immune checkpoint inhibitors (ICIs), which stimulate antitumor immunity, have also demonstrated a remarkable clinical benefit in the treatment of many aggressive malignancies. Nevertheless, the emergence of resistance to immunotherapies associated with an immunosuppressive tumor microenvironment has restricted therapeutic response, necessitating the development of better therapeutic strategies to increase treatment efficacy in patients. Angiopoietin-2 (ANG2), which binds to the receptor tyrosine kinase TIE2 in endothelial cells, is a cooperative driver of angiogenesis and vascular destabilization along with VEGF. It has been suggested in multiple preclinical studies that ANG2-mediated vascular changes contribute to the development and persistence of resistance to anti-VEGF therapy. Further, emerging evidence suggests a fundamental link between vascular abnormalities and tumor immune evasion, supporting the rationale for combination strategies of immunotherapy with antiangiogenic drugs. In this review, we discuss the recent mechanistic and clinical advances in targeting angiopoietin signaling, focusing on ANG2 inhibition, to enhance therapeutic efficacy of antiangiogenic and ICI therapies. In short, we propose that a better mechanistic understanding of ANG2-mediated vascular changes will provide insight into the significance of ANG2 in treatment response and resistance to current antiangiogenic and ICI therapies. These advances will ultimately improve therapeutic modalities for cancer treatment.

Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi-tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c-Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro-angiogenic macrophage subset, Tie2-expressing macrophages (TEM). Angi-Tie2 and EphB4-EphrinB2 signaling plays critical roles in pericyte-mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well-tolerated combination of multi-targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

To investigate the intervention therapy effect on the growth of malignant melanoma, we have made an observation of expression levels of Ang2 in malignant melanoma cells, which was transduced by small interfering RNA (Ang2-siRNA) of Ang2 in vitro and in vivo. We successfully constructed Ang2-siRNA lent virus, and constructed nude mice model by transplanting malignant melanoma. Ang2-siRNA lent virus inhibited Ang2 mRNA of malignant melanoma in vitro and in vivo, and inhibited malignant melanoma tumor growth at the same time. Ang2-siRNA lent virus can interfere expression levels of Ang2 in malignant melanoma cells, inhibit tumor growth, and silent Ang2 gene expression, which may pave a new way for clinical gene therapy of malignant melanoma.

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively The degree of irt vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic factors produced by AML cells. The expression of different angiogenic factors was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial growth factor (VEGF) expression, 4/19 expressed varied spontaneous blastic fibroblast growth factor mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/ or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the growth advantage of the malignant counterpart as a result of the paracrine production of growth factors produced by the surrounding endothelial cells.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively The degree of irt vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic factors produced by AML cells. The expression of different angiogenic factors was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial growth factor (VEGF) expression, 4/19 expressed varied spontaneous blastic fibroblast growth factor mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/ or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the growth advantage of the malignant counterpart as a result of the paracrine production of growth factors produced by the surrounding endothelial cells.

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2−/− mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2−/− mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2−/− pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2−/− mice and further defines the sequential steps in lymphatic vessel remodeling.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively The degree of irt vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic factors produced by AML cells. The expression of different angiogenic factors was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial growth factor (VEGF) expression, 4/19 expressed varied spontaneous blastic fibroblast growth factor mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/ or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the growth advantage of the malignant counterpart as a result of the paracrine production of growth factors produced by the surrounding endothelial cells.

Lung cancer is the leading cause of cancer-associated deaths worldwide. Platinum-based chemotherapy is the most common therapeutic approach in advanced non-small cell lung cancer (NSCLC), particularly for tumors that carry non-druggable activating mutations. However, these tumors are generally not eradicated when diagnosed at an advanced stage and are frequently resistant to chemotherapy. Therefore, there is an urgent medical need for new treatments for this frequent cancer type. Immunotherapies are treatments based on increasing the strength of immune responses against cancer. In particular, monoclonal antibodies that block inhibitory "immune-checkpoints" expressed on T cells (e.g., programmed cell death protein 1, or PD1) show clinical efficacy in increasing tumor types. However, these therapies improve survival in only a minority of NSCLC patients, and ongoing efforts are being made for increasing their efficacy through combinatorial treatments. In this regard, growing evidence indicates that tumor angiogenesis is closely associated with immunosuppression. Pro-angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANG2), cause abnormalities and dysfunctions in tumor blood vessels that impair T cell extravasation and anti-tumoral activity. Recent findings in preclinical cancer models show that reprogramming the features of tumor-associated blood vessels by anti-angiogenic drugs may help to increase intratumoral T cell abundance and sensitize refractory tumors to immunotherapy. Nevertheless, the potential of anti-angiogenics in the context of lung cancer immunotherapy is poorly understood. Therefore, the main objective of my project was to audit combinations of anti-angiogenic therapy and immune checkpoint blockade in a genetically engineered mouse model of NSCLC. I first studied the anti-tumoral activity of dual VEGFA and ANG2 inhibition using a bispecific antibody (A2V) in the KrasLSL-G12D/+;p53fl/fl (KP) NSCLC model. I found that A2V, but not single VEGFA or ANG2 blockade, had robust tumor-inhibitory activity in KP mice, which was at least comparable to standard-of-care chemotherapy. However, A2V only moderately increased cytotoxic T cells in the tumors. To potentially increase the immunogenicity of KP tumors, I also generated two KP mouse variants by either co-expressing a surrogate neoantigen or genetically deleting a DNA mismatch repair enzyme in the cancer cells. However, the therapeutic efficacy of A2V was not further improved in spite of a slightly more activated immune response. I next investigated combination therapies involving A2V and PD1 blockade. In contrast to previous findings in other cancer types, the addition of PD1 antibodies deteriorated the efficacy of A2V, likely through enhancing immunosuppressive mechanisms sustained, at least in part, by regulatory T cells and T cell-antagonizing cytokines. In summary, my work supports the potential clinical efficacy of combined VEGFA and ANG2 blockade for lung cancer therapy but provided little evidence for its synergy in association with anti-PD1 therapy.