Anti-angiogenic Strategies Research Articles

Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles.

Existing vascular endothelial growth factor-oriented antiangiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel antiangiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31-targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters, and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. SIGNIFICANCE STATEMENT: Systemic administration of antiangiogenic therapeutics induces side effects to non-targeted tissues. This study, among others, has shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, this study generated CD31-targeting liposomes with encapsulated Fasudil, a clinically relevant Rho kinase inhibitor, and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations, and angiogenic functions verify the benefits of site-targeted antiangiogenic therapy.

Extracellular Vesicles as Delivery Shippers for Noncoding RNA-Based Modulation of Angiogenesis: Insights from Ischemic Stroke and Cancer.

Ischemic stroke and systemic cancer are two of the leading causes of mortality. Hypoxia is a central pathophysiological component in ischemic stroke and cancer, representing a joint medical function. This function includes angiogenesis regulation. Vascular remodeling coupled with axonal outgrowth following cerebral ischemia is critical in improving poststroke neurological functional recovery. Antiangiogenic strategies can inhibit cancer vascularization and play a vital role in impeding cancer growth, invasion, and metastasis. Although there are significant differences in the cause of angiogenesis across both pathophysiological conditions, emerging evidence states that common signaling structures, such as extracellular vesicles (EVs) and noncoding RNAs (ncRNAs), are involved in this context. EVs, heterogeneous membrane vesicles encapsulating proteomic genetic information from parental cells, act as multifunctional regulators of intercellular communication. Among the multifaceted roles in modulating biological responses, exhaustive evidence shows that ncRNAs are selectively sorted into EVs, modulating common specific aspects of cancer development and stroke prognosis, namely, angiogenesis. This review will discuss recent advancements in the EV-facilitated/inhibited progression of specific elements of angiogenesis with a particular concern about ncRNAs within these vesicles. The review is concluded by underlining the clinical opportunities of EV-derived ncRNAs as diagnostic, prognostic, and therapeutic agents.

A novel risk score model based on four angiogenesis long non-coding RNAs for prognosis evaluation of pancreatic adenocarcinoma.

Long non-coding RNAs (lncRNAs) have been reported to play significant roles in tumour angiogenesis which prominently facilitates pancreatic adenocarcinoma (PAAD) progression. The clinical PAAD data were obtained from TCGA database and clinical specimens of 122 PAAD patients. The Molecular Signatures Database v4.0 was used to identify angiogenesis-related long non-coding RNAs (ARLNRs). Survival-related ARLNRs (sARLNRs) were further validated by univariate and multivariate COX regression analyses. The expressions of CASC8, AC015660.1, Z97832.2 and PAN3-AS1 in PAAD cell lines and tissues were examined by qPCR. The correlations between sARLNRs (CASC8 and AC015660.1) and clinicopathological characteristics of the 122 PAAD patients were analyzed by the chi-square test and Fisher's exact probability method. 590 lncRNAs were identified as ARLNRs, of which four sARLNRs were further used to establish an angiogenesis-related risk score model (ARRS), by which patients in the low-risk group have better survival probabilities than those in the high-risk group. The expression levels of CASC8 and AC015660.1 were significantly higher in PAAD cell lines and tumor tissues especially in patients with advanced grades and T-stages, while Z97832.2 and PAN3-AS1 were inverse. In addition, the higher expression of CASC8 and AC015660.1 prominently associated with the larger tumour size, and the more advanced grade and T-stage. However, the relevance between the sARLNRs (CASC8 and AC015660.1) expression and lymph node metastasis status was not significant. In the study, we illuminate the clinical significance, angiogenesis relevance and prognosis-predictive value of four sARLNRs for PAAD. The results build a bridge between sARLNRs and tumour vascularization, and also establish a reliable and accurate risk scoring model for PAAD antiangiogenic strategy.

Manipulation of the crosstalk between tumor angiogenesis and immunosuppression in the tumor microenvironment: Insight into the combination therapy of anti-angiogenesis and immune checkpoint blockade.

Immunotherapy has been recognized as an effective and important therapeutic modality for multiple types of cancer. Nevertheless, it has been increasing recognized that clinical benefits of immunotherapy are less than expected as evidenced by the fact that only a small population of cancer patients respond favorably to immunotherapy. The structurally and functionally abnormal tumor vasculature is a hallmark of most solid tumors and contributes to an immunosuppressive microenvironment, which poses a major challenge to immunotherapy. In turn, multiple immune cell subsets have profound consequences on promoting neovascularization. Vascular normalization, a promising anti-angiogenic strategy, can enhance vascular perfusion and promote the infiltration of immune effector cells into tumors via correcting aberrant tumor blood vessels, resulting in the potentiation of immunotherapy. More interestingly, immunotherapies are prone to boost the efficacy of various anti-angiogenic therapies and/or promote the morphological and functional alterations in tumor vasculature. Therefore, immune reprograming and vascular normalization appear to be reciprocally regulated. In this review, we mainly summarize how tumor vasculature propels an immunosuppressive phenotype and how innate and adaptive immune cells modulate angiogenesis during tumor progression. We further highlight recent advances of anti-angiogenic immunotherapies in preclinical and clinical settings to solidify the concept that targeting both tumor blood vessels and immune suppressive cells provides an efficacious approach for the treatment of cancer.

Classical Angiogenic Signaling Pathways and Novel Anti-Angiogenic Strategies for Colorectal Cancer.

Although productive progress has been made in colorectal cancer (CRC) researchs, CRC is the second most frequent type of malignancy and the major cause of cancer-related death among gastrointestinal cancers. As angiogenesis constitutes an important point in the control of CRC progression and metastasis, understanding the key signaling pathways that regulate CRC angiogenesis is critical in elucidating ways to inhibit CRC. Herein, we comprehensively summarized the angiogenesis-related pathways of CRC, including vascular endothelial growth factor (VEGF), nuclear factor-kappa B (NF-κB), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), Wingless and int-1 (Wnt), and Notch signaling pathways. We divided the factors influencing the specific pathway into promoters and inhibitors. Among these, some drugs or natural compounds that have antiangiogenic effects were emphasized. Furthermore, the interactions of these pathways in angiogenesis were discussed. The current review provides a comprehensive overview of the key signaling pathways that are involved in the angiogenesis of CRC and contributes to the new anti-angiogenic strategies for CRC.

A Novel Artificial Intelligence-Based Approach for Quantitative Assessment of Angiogenesis in the Ex Ovo CAM Model.

Simple SummaryAngiogenesis is crucial in tissue regeneration and a relevant factor in tumor growth. Consequently, there is a demand for methods to assess angiogenesis. The chorioallantoic membrane (CAM) assay is a widely used in vivo model for the study of angiogenesis. However, there is no established gold standard to evaluate the vascularisation of the CAM, which limits the comparability of the different studies. In this manuscript, we present a novel approach to address this topic.Angiogenesis is a highly regulated process. It promotes tissue regeneration and contributes to tumor growth. Existing therapeutic concepts interfere with different steps of angiogenesis. The quantification of the vasculature is of crucial importance for research on angiogenetic effects. The chorioallantoic membrane (CAM) assay is widely used in the study of angiogenesis. Ex ovo cultured chick embryos develop an easily accessible, highly vascularised membrane on the surface. Tumor xenografts can be incubated on this membrane enabling studies on cancer angiogenesis and other major hallmarks. However, there is no commonly accepted gold standard for the quantification of the vasculature of the CAM. We compared four widely used measurement techniques to identify the most appropriate one for the quantification of the vascular network of the CAM. The comparison of the different quantification methods suggested that the CAM assay application on the IKOSA platform is the most suitable image analysis application for the vasculature of the CAM. The new CAM application on the IKOSA platform turned out to be a reliable and feasible tool for practical use in angiogenesis research. This novel image analysis software enables a deeper exploration of various aspects of angiogenesis and might support future research on new anti-angiogenic strategies for cancer treatment.

Downregulation of Manic fringe impedes angiogenesis and cell migration of renal carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a highly angiogenic cancer. Manic fringe (MFng) is elevated in ccRCC compared to the normal kidney. However, its role in ccRCC tumour angiogenesis remains elusive. This study seeks to determine the expression pattern of MFng in ccRCC blood vessels and its role in angiogenesis. The association between MFng and the blood vessels was established through online compendia, immunohistochemistry and qPCR analyses. The anti-angiogenic potential of lentiviral-mediated MFng knockdown in endothelial cells (EC shMFng) was assessed for viability, proliferation, apoptosis, migration, adhesion, cell cycle, vessel sprouting, and molecular expression of adhesion and apoptosis markers. Finally, EC shMFng were co-cultured with 786-0 renal cancer cells to determine their impact on cancer cell migration. The online dataset analyses and immunostaining on ccRCC tissues revealed high expression of MFng in ECs. MFng and CD31/PECAM-1 genes were up-regulated in ccRCC tissue samples compared to normal kidney tissues. EC shMFng demonstrated decreased cell viability due to G1 cell cycle arrest and reduced Ki-67 protein expression. In addition, shMFng down-regulated endothelial adhesion molecules and hindered EC migration, network formation and sprouting, compared to their respective empty vector (EV) controls. Co-culture assay of EC shMFng with 786-0 renal cancer cells inhibited cancer cell migration. These findings underscore the potential role of MFng in ECs in influencing renal cancer cell migration, thus opening an avenue for anti-angiogenic strategy targeting MFng to treat ccRCC.

Research Progress on Angiogenesis Imbalance and Pathogenesis of Preeclampsia and Predictive Treatment

Preeclampsia, one of the unique diseases of pregnancy, is a systemic vascular disease induced by various factors. Its main feature is that new-onset hypertension and proteinuria will appear after 20 weeks of pregnancy. Preeclampsia is the main cause of morbidity and mortality in pregnant women and perinatal babies. Many clinical and experimental studies have shownthat the pathological basis of preeclampsia is maternal endothelial dysfunction caused by placental factors. Moreover, it has been determined that the increase in placental anti-angiogenic factors is the main cause of vascular endothelial dysfunction and systemic vascular dysfunction in pregnant women. This review summarizes the latest advancesin the molecular mechanisms of endothelial dysfunction caused by placental anti-angiogenic factors and new clinical strategies based on these findings.

Novel Drugs with High Efficacy against Tumor Angiogenesis.

Angiogenesis is involved in physiological and pathological processes in the body. Tumor angiogenesis is a key factor associated with tumor growth, progression, and metastasis. Therefore, there is great interest in developing antiangiogenic strategies. Hypoxia is the basic initiating factor of tumor angiogenesis, which leads to the increase of vascular endothelial growth factor (VEGF), angiopoietin (Ang), hypoxia-inducible factor (HIF-1), etc. in hypoxic cells. The pathways of VEGF and Ang are considered to be critical steps in tumor angiogenesis. A number of antiangiogenic drugs targeting VEGF/VEGFR (VEGF receptor) or ANG/Tie2, or both, are currently being used for cancer treatment, or are still in various stages of clinical development or preclinical evaluation. This article aims to review the mechanisms of angiogenesis and tumor angiogenesis and to focus on new drugs and strategies for the treatment of antiangiogenesis. However, antitumor angiogenic drugs alone may not be sufficient to eradicate tumors. The molecular chaperone heat shock protein 90 (HSP90) is considered a promising molecular target. The VEGFR system and its downstream signaling molecules depend on the function of HSP90. This article also briefly introduces the role of HSP90 in angiogenesis and some HSP90 inhibitors.

Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy

Anti-angiogenic cancer therapies possess immune-stimulatory properties by counteracting pro-angiogenic molecular mechanisms. We report that tumor endothelial cells ubiquitously overexpress and secrete the intermediate filament protein vimentin through type III unconventional secretion mechanisms. Extracellular vimentin is pro-angiogenic and functionally mimics VEGF action, while concomitantly acting as inhibitor of leukocyte-endothelial interactions. Antibody targeting of extracellular vimentin shows inhibition of angiogenesis in vitro and in vivo. Effective and safe inhibition of angiogenesis and tumor growth in several preclinical and clinical studies is demonstrated using a vaccination strategy against extracellular vimentin. Targeting vimentin induces a pro-inflammatory condition in the tumor, exemplified by induction of the endothelial adhesion molecule ICAM1, suppression of PD-L1, and altered immune cell profiles. Our findings show that extracellular vimentin contributes to immune suppression and functions as a vascular immune checkpoint molecule. Targeting of extracellular vimentin presents therefore an anti-angiogenic immunotherapy strategy against cancer.

A two-dimensional mathematical model of tumor angiogenesis with CD147.

Tumor angiogenesis is the tumor's inherent blood supply system which is crucial for the growth of tumor. Extracellular Matrix Metallo Proteinases Inducer (EMMPRIN)/Cluster of Differentiation 147 (CD147) is found in high levels on tumor surfaces. This study focuses on these elevated levels of CD147 and the effect it has on tumor angiogenesis. The present article develops a Two-Dimensional Mathematical Model of Tumor Angiogenesis taking into account the CD147 molecule. The effects of CD147 on Tumor Angiogenesis Factors (TAFs), fibronectin and Matrix Metallo Proteinases (MMPs) are also incorporated. The results have been obtained through COMSOL Multiphysics 5.4 software. The results show that CD147 is responsible for swifter angiogenesis, calling for targeting this molecule in anti-angiogenic strategies. The present model is validated with the existing theoretical and experimental results.

Cyclic-AMP signalling, MYC and hypoxia-inducible factor 1α intersect to regulate angiogenesis in B-cell lymphoma.

Angiogenesis and MYC expression associate with poor outcome in diffuse large B-cell lymphoma (DLBCL). MYC promotes neo-vasculature development but whether its deregulation in DLBCL contributes to angiogenesis is unclear. Examination of this relationship may uncover novel pathogenic regulatory circuitry as well as anti-angiogenic strategies in DLBCL. Here, we show that MYC expression positively correlates with vascular endothelial growth factor (VEGF) expression and angiogenesis in primary DLBCL biopsies, independently of dual expressor status or cell-of-origin classification. We found that MYC promotes VEGFA expression, a correlation that was validated in large datasets of mature B-cell tumours. Using DLBCL cell lines and patient-derived xenograft models, we identified the second messenger cyclic-AMP (cAMP) as a potent suppressor of MYC expression, VEGFA secretion and angiogenesis in DLBCL in normoxia. In hypoxia, cAMP switched targets and suppressed hypoxia-inducible factor 1α, a master regulator of VEGFA/angiogenesis in low oxygen environments. Lastly, we used the phosphodiesterase 4b (Pde4b) knockout mouse to demonstrate that the cAMP/PDE4 axis exercises additional anti-angiogenesis by directly targeting the lymphoma microenvironment. In conclusion, MYC could play a direct role in DLBCL angiogenesis, and modulation of cAMP levels, which can be achieved with clinical grade PDE4 inhibitors, has cell and non-cell autonomous anti-angiogenic activity in DLBCL.

Antiangiogenic Strategies in Epithelial Ovarian Cancer: Mechanism, Resistance, and Combination Therapy.

Angiogenesis is one of the hallmarks of cancer and plays a crucial role in carcinogenesis and progression of epithelial ovarian cancer. Antiangiogenic agent is the first approved targeted agent in ovarian cancer. Anti-angiogenic agents mainly include agents target VEGF/VEGFR pathway, such as bevacizumab and agents target receptor tyrosine kinase, and non-VEGF/VEGFR targets of angiogenesis. Antiangiogenic agents demonstrate certain effects in ovarian cancer treatment either as monotherapy or combined with chemotherapy. Unfortunately, antiangiogenic agents, such as bevacizumab, integrated into the ovarian cancer treatment paradigm do not increase cures. Thus, the benefits of anti-angiogenic agents must be carefully weighed against the cost and associated toxicities. Antiangiogenic agents drug resistance and short of predictive biomarkers are main obstacles in ovarian cancer treatment. A combination of poly (ADP-ribose) polymerase inhibitors or immune checkpoint inhibitors might be great strategies to overcome resistance as well as enhance anti-tumor activity of anti-angiogenic drugs. Predictive biomarkers of antiangiogenic agents are in urgent need.

Synthesis and biological evaluation of marine natural product, Cryptoechinuline D derivatives as novel antiangiogenic agents

Tumor angiogenesis is an important biological process involved in the proliferation and migration of endothelial cells, regulated by Ang/Tie-2 signaling pathways, which is essential for tumor growth and metastasis. Therefore, blocking Ang/Tie-2 signaling pathways is a promising anti-angiogenic strategy for tumor treatment. 2,5-Diketopiperazines (DKPs) are a kind of bioactive compounds derived from marine fungi and they present a wide spectrum of pharmacological properties, particularly in the field of cancer treatment. Herein, a DKP marine natural product, Cryptoechinuline D (Cry D) was applied to structural modification and twelve derivatives were synthesized. Among which, compound 5 showed significant inhibitory activity against HUVECs with an IC50 value of 12.6 μmol/L, which weakened the proliferation, migration and invasion of HUVECs by inhibiting the Ang2/Tie-2 signaling pathway. The results of these evaluations indicated that compound 5 might be a promising anti-angiogeneic agent and worth further optimization and development for cancer therapy.

Vascular Endothelial Growth Factor Receptors [VEGFR] as Target in Breast Cancer Treatment: Current Status in Preclinical and Clinical Studies and Future Directions.

Breast cancer [BC] is one of the most common cancers among women, one of the leading causes of a considerable number of cancer-related death globally. Among all procedures leading to the formation of breast tumors, angiogenesis has an important role in cancer progression and outcomes. Therefore, various anti-angiogenic strategies have been developed so far to enhance treatment's efficacy in different types of BC. Vascular endothelial growth factors [VEGFs] and their receptors are regarded as the most well-known regulators of neovascularization. VEGF binding to vascular endothelial growth factor receptors [VEGFRs] provides cell proliferation and vascular tissue formation by the subsequent tyrosine kinase pathway. VEGF/VEGFR axis displays an attractive target for anti-angiogenesis and anti-cancer drug design. This review aims to describe the existing literature regarding VEGFR inhibitors, focusing on BC treatment reported in the last two decades.

EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities.

Simple SummaryTumor angiogenesis is an important step in the progression of solid tumors. Understanding the mechanisms involved in tumor vasculature formation is critical for improving anti-angiogenic strategies. In this study, we reported that EGFR mutation-containing lung cancer cells produced CLEC11A with endothelial trophic and tumor-promoting activities. CLEC11A could be a novel factor involved in tumor angiogenesis.The formation of new blood vessels in solid tumors is regulated by various endothelial trophic factors. We identified that CLEC11A, an extracellular C-type lectin, was over-expressed in lung cancer cell lines harboring mutated EGFR. CLEC11A expression was also frequently elevated in lung adenocarcinoma (LAC) tissues with EGFR mutation. CLEC11A-expressing H1299 cells formed larger tumors in nude mice than did the control cells. The CLEC11A-expressing tumors contained more CD31-positive cells, suggesting that they had a higher angiogenic activity. CLEC11A per se did not induce blood vessel formation, but enhanced angiogenesis triggered by VEGF-A or basic FGF in vivo. Additionally, the expression of small hairpin RNA against CLEC11A (shCLEC11A) in HCC827 LAC cells suppressed their tumorigenic ability. Purified CLEC11A exhibited a chemotactic ability, which is dependent on its integrin-binding RGD and LDT motifs, toward endothelial cells. This chemotactic activity was not affected by the presence of a VEGFR inhibitor. Conditioned medium produced by HCC827-shCLEC11A cells had diminished chemotactic ability toward endothelial cells. CLEC11A treatments increased the levels of active integrin β1 that were not associated with activation of focal adhesion kinases in endothelial cells. Our results indicated that CLEC11A was a factor of angiogenic potential and was involved in lung cancer tumorigenesis.

Myricetin reverses epithelial-endothelial transition and inhibits vasculogenic mimicry and angiogenesis of hepatocellular carcinoma by directly targeting PAR1.

Most antiangiogenic inhibitors targeting endothelium-dependent vessels cannot inhibit tumor growth but promote tumor invasion and metastasis in some patients. Vasculogenic mimicry (VM) employs mechanisms that differ from those used to construct endothelium-dependent vessels. Inhibiting VM may be a novel antiangiogenic strategy against alternative tumor vascularization. In this paper, myricetin was selected from among several flavonoid compounds as an effective PAR1 antagonist. In two different hepatocellular carcinoma (HCC) cell lines high-expressed PAR1, myricetin inhibited cell migration, invasion and VM formation and reversed the expression of epithelial-endothelial transition (EET) markers by inhibiting PAR1 activation. Knockout of PAR1 inhibited HCC cell invasion and metastasis and weakened the inhibitory effect of myricetin on HCC cells. The migration, invasion and tube formation ability of PLC-PRF-5 cells were enhanced after PAR1 overexpression, and the inhibitory effect of myricetin was enhanced. A docking assay revealed that myricetin binds to Leu258 and Thr261 in the PAR1 activity pocket. Mutation of Leu258 and Thr261 inhibited the antitumor effect of myricetin in vitro and in vivo. In summary, myricetin reverses PAR1-mediated EET and inhibits HCC cell invasion, metastasis, VM formation and angiogenesis by targeting PAR1, and Leu258 and Thr261 of PAR1 participate in VM and angiogenesis in HCC tissues.

Docetaxel and 5-FU enhanced the inhibitory effects of apatinib and ramucirumab on growth and migration of gastric cancer

BackgroundGastric cancer (GC) is one of the most common malignant tumors in the world. The clinical benefit of anti-angiogenic strategy as a single drug is limited. Some studies showed that the combination of anti-angiogenic therapy and chemotherapy exhibited synergistic effect and reduced the side effects of chemotherapy drugs. We investigated the combined effects of these two types of drugs in gastric cancer cells in vitro and in vivo. Methodscell viability, migration, invasion, and apoptosis were evaluated by CCK-8, wound-healing, transwell, and Annexin V-FITC/PI assay, respectively. In vivo anti-cancer efficacy was tested for the cell proliferation and metastasis in cell line derived tumor xenograft (CDX) model and patient derived tumor xenografted (PDX) model based on Tg (fli-1: EGFP) zebrafish embryos; ResultsIn the cell experiments, the combination of the two types of drugs could inhibit the proliferation and metastasis of gastric cancer cells and promote apoptosis through VEGFR-2/AKT/ERK1/2 signal. In the zebrafish CDX (zCDX) model and zebrafish PDX (zPDX) model, the combination of the two treatment also showed a synergistic effect in inhibiting gastric cancer cell metastasis and cell proliferation. ConclusionsApatinib/ramucirumab targeted therapy combined with docetaxel or 5-fluorouracil (5-FU) may serve as an effective treatment strategy for patients with advanced gastric cancer.

A New Approach to Inhibiting Triple-Negative Breast Cancer: In Vitro, Ex Vivo and In Vivo Antiangiogenic Effect of BthTx-II, a PLA2-Asp-49 from Bothrops jararacussu Venom.

Phospholipases A2 (PLA2) represent a superfamily of enzymes widely distributed in living organisms, with a broad spectrum of pharmacological activities and therapeutic potential. Anti-angiogenic strategies have become one of the main tools in fighting cancer. In this sense, the present work reports the inhibition of tumor angiogenesis induced by Asp-49 BthTX-II using in vitro, ex vivo and in vivo approaches. We demonstrate that BthTx-II inhibited cell adhesion, proliferation, and migration of human umbilical vein endothelial cells (HUVEC), as well as caused a reduction in the levels of endothelial growth factor (VEGF) during in vitro angiogenesis assays. BthTx-II was also able to inhibit the sprouting angiogenic process, by the ex vivo germination assay of the aortic ring; in addition, this toxin inhibited the migration and proliferation of HUVEC in co-culture with triple-negative breast cancer cells (e.g., MDA-MB-231 cells). Finally, in vivo tumor suppression and anti-angiogenic activities were analyzed using MDA-MB-231 cells with Matrigel injected into the chorioallantoic membrane of chicken embryo (CAM) for 7 days treatment with BthTx-II, showing a considerable reduction in vessel caliber, on the size and weight of tumors. Together, these results suggest an important antiangiogenic and antitumor role for BthTx-II, as a potential prototype for the development of new tools and antitumor drugs in cancer therapy.

Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.

Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated Tcells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.