Nonsynonymous mutations in VEGF receptor binding domain alter the efficacy of bevacizumab treatment.

Vascular endothelial growth factor (VEGF) mediated angiogenesis is crucial for tumor progression. Isoforms of VEGF bind to different VEGF receptors (VEGFRs) to initiate angiogenesis specific cellular signaling. Inhibitors that target both the receptors and ligands are in clinical use to impede angiogenesis. Bevacizumab, a monoclonal antibody (mAb) approved by the Food and Drug Administration (FDA), binds in the VEGF receptor binding domain (RBD) of all soluble isoforms of VEGF and inhibits the VEGF-VEGFR interaction. Bevacizumab is also used in combination with other chemotherapeutic agents for a better therapeutic outcome. Understanding the intricate polymorphic character of VEGFA gene and the influence of missense or nonsynonymous mutations in the form of nonsynonymous polymorphisms (nsSNPs) on RBD of VEGF may aid in increasing the efficacy of this drug. This study has identified 18 potential nsSNPs in VEGFA gene that affect the VEGF RBD structure and alter its binding pattern to bevacizumab. The mutated RBDs, modeled using trRosetta, in addition to the changed pattern of secondary structure, post translational modification and stability compared to the wild type, have shown contrasting binding affinity and molecular interaction pattern with bevacizumab. Molecular docking analysis by ClusPro and visualization using PyMol and PDBsum tools have detected 17 nsSNPs with decreased binding affinity to bevacizumab and therefore may impact the treatment efficacy. Whereas VEGF RBD expressed due to rs1267535717 (R229H) nsSNP of VEGFA has increased affinity to the mAb. This study suggests that genetic characterization of VEGFA before bevacizumab mediated cancer treatment is essential in predicting the appropriate efficacy of the drug, as the treatment efficiency may vary at individual level.

IL-36γ Augments Ocular Angiogenesis by Promoting the Vascular Endothelial Growth Factor-Vascular Endothelial Growth Factor Receptor Axis.

Prevention of inflammatory angiogenesis is critical for suppressing chronic inflammation and inhibiting inflammatory tissue damage. Angiogenesis is particularly detrimental to the cornea because pathologic growth of new blood vessels can lead to marked vision impairment and even loss of vision. The expression of proinflammatory cytokines by injured tissues exacerbates the inflammatory cascade, including angiogenesis. IL-36 cytokine, a subfamily of the IL-1 superfamily, consists of three proinflammatory agonists, IL-36α, IL-36ß, and IL-36γ, and an IL-36 receptor antagonist (IL-36Ra). Data from the current study indicate that human vascular endothelial cells constitutively expressed the cognate IL-36 receptor. The current investigation, for the first time, characterized the direct contribution of IL-36γ to various angiogenic processes. IL-36γ up-regulated the expression of vascular endothelial growth factors (VEGFs) and their receptors VEGFR2 and VEGFR3 by human vascular endothelial cells, suggesting that IL-36γ mediates the VEGF-VEGFR signaling by endothelial cells. Moreover, by using a naturally occurring antagonist IL-36Ra in a murine model of inflammatory angiogenesis, this study demonstrated that blockade of endogenous IL-36γ signaling results in significant retardation of inflammatory angiogenesis. The current investigation on the proangiogenic function of IL-36γ provides novel evidence of the development of IL-36γ-targeting strategies to hamper inflammatory angiogenesis.

Interplay of vascular endothelial growth factor receptors in organ-specific vessel maintenance.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are quintessential for the development and maintenance of blood and lymphatic vessels. However, genetic interactions between the VEGFRs are poorly understood. VEGFR2 is the dominant receptor that is required for the growth and survival of the endothelium, whereas deletion of VEGFR1 or VEGFR3 was reported to induce vasculature overgrowth. Here we show that vascular regression induced by VEGFR2 deletion in postnatal and adult mice is aggravated by additional deletion of VEGFR1 or VEGFR3 in the intestine, kidney, and pancreas, but not in the liver or kidney glomeruli. In the adult mice, hepatic and intestinal vessels regressed within a few days after gene deletion, whereas vessels in skin and retina remained stable for at least four weeks. Our results show changes in endothelial transcriptomes and organ-specific vessel maintenance mechanisms that are dependent on VEGFR signaling pathways and reveal previously unknown functions of VEGFR1 and VEGFR3 in endothelial cells.

Rapamycin as a potent and selective inhibitor of vascular endothelial growth factor receptor in breast carcinoma.

Angiogenesis is the process of new vascular formation, which is derived from various factors. For suppressing cancer cell growth, targeting angiogenesis is one of the therapeutic approaches. Vascular endothelial growth factor family receptors, including Flt-1, Flk-1 and Flt-4, have been found to play an essential role in regulating angiogenesis. Rapamycin is a macrolide compound with anti-proliferative properties, while platelet factor-4 (PF-4) is an antiangiogenic ELR-negative chemokine. Rapamycin inhibits mTOR ligands activation, thus suppressing cell proliferation, while PF-4 inhibits cell proliferation through several mechanisms. In the present study, we evaluated the effects of rapamycin and platelet factor-4 toward breast carcinoma at the proteomic and genomic levels. A total of 60 N-Methyl-N-Nitrosourea-induced rat breast carcinomas were treated with rapamycin, platelet factor-4 and rapamycin+platelet factor-4. The tumours were subsequently subjected to immunohistochemical protein analysis and polymerase chain reaction gene analysis. Protein analysis was performed using a semiquantitative scoring method, while the mRNA expression levels were analysed based on the relative expression ratio. There was a significant difference in the protein and mRNA expression levels for the selected markers. In the rapamycin+platelet factor-4-treated group, the Flt-4 marker was downregulated, whereas there were no differences in the expression levels of other markers, such as Flt-1 and Flk-1. On the other hand, platelet factor-4 did not exhibit a superior angiogenic inhibiting ability in this study. Rapamycin is a potent antiangiogenic drug; however, platelet factor-4 proved to be a less effective drug of anti-angiogenesis on rat breast carcinoma model.

VEGF-A, VEGFR1 and VEGFR2 single nucleotide polymorphisms and outcomes from the AGITG MAX trial of capecitabine, bevacizumab and mitomycin C in metastatic colorectal cancer.

The phase III MAX clinical trial randomised patients with metastatic colorectal cancer (mCRC) to receive first-line capecitabine chemotherapy alone or in combination with the anti-VEGF-A antibody bevacizumab (± mitomycin C). We utilised this cohort to examine whether single nucleotide polymorphisms (SNPs) in VEGF-A, VEGFR1, and VEGFR2 are predictive of efficacy outcomes with bevacizumab or the development of hypertension. Genomic DNA extracted from archival FFPE tissue for 325 patients (69% of the MAX trial population) was used to genotype 16 candidate SNPs in VEGF-A, VEGFR1, and VEGFR2, which were analysed for associations with efficacy outcomes and hypertension. The VEGF-A rs25648 'CC' genotype was prognostic for improved PFS (HR 0.65, 95% CI 0.49 to 0.85; P = 0.002) and OS (HR 0.70, 95% CI 0.52 to 0.94; P = 0.019). The VEGF-A rs699947 'AA' genotype was prognostic for shorter PFS (HR 1.32, 95% CI 1.002 to 1.74; P = 0.048). None of the analysed SNPs were predictive of bevacizumab efficacy outcomes. VEGFR2 rs11133360 'TT' was associated with a lower risk of grade ≥ 3 hypertension (P = 0.028). SNPs in VEGF-A, VEGFR1 and VEGFR2 did not predict bevacizumab benefit. However, VEGF-A rs25648 and rs699947 were identified as novel prognostic biomarkers and VEGFR2 rs11133360 was associated with less grade ≥ 3 hypertension.

Vascular heterogeneity: VEGF receptors make blood vessels special.

Karaman et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20210565) examined the differential effects of the conditional deletion of genes encoding each VEGF receptor, VEGFR1, VEGFR2 and VEGFR 3, as well as combinations thereof in mice. The results highlight the crosstalk between receptors in different organs and emphasize the importance of VEGF receptor expression and interplay in vascular heterogeneity.

Salvianolic acid B and ferulic acid synergistically promote angiogenesis in HUVECs and zebrafish via regulating VEGF signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Induced vascular growth in the myocardium has been widely acknowledged as a promising intervention strategy for patients with ischemic coronary artery disease. Yet despite long-term efforts on gene, protein or cell-based pro-angiogenic therapies, the clinical translation remains challenging. Noticeably, multiple medicinal herbs have long-term documented effects in promoting blood circulation. Salvia miltiorrhiza and Ligusticum stratum are two representative traditional Chinese medicine herbs with suggested roles in enhancing organ blood supply, and Guanxinning Tablet (GXNT), a botanical drug which is formulated with these two herbs, exhibited significant efficacy against angina pectoris in clinical practices. AIM OF THE STUDY: This study aimed to examine the pro-angiogenic activity of GXNT and its major components, as well as to explore their pharmacological mechanism in promoting angiogenesis. MATERIALS AND METHODS: In vitro, the pro-angiogenic effects of GXNT and its major components were examined on human umbilical vein endothelial cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch assay, and endothelial cell tube formation assay. In vivo, the pro-angiogenic effects were examined on the ponatinib-induced angiogenesis defective zebrafish model. The active compounds were identified through phenotype-based screening in zebrafish, and their pharmacological mechanism was explored in both in vitro and in vivo models by immunofluorescent staining, cell cycle analysis, quantitative PCR and whole embryo in-situ hybridization. RESULTS: We demonstrated strong pro-angiogenic effects of GXNT in both human umbilical vein endothelial cells and zebrafish model. Moreover, through phenotype-based screening in zebrafish for active compounds, pro-angiogenic effects was discovered for salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, and its activity was further enhanced when co-administered with ferulic acid (FA), which is contained in Ligusticum stratum. On the cellular level, Sal B and FA cotreatment increased endothelial cell proliferation of sprouting arterial intersomitic vessels in zebrafish, as well as largely restored G1-S cell cycle progression and cyclin D1 expression in angiogenic defective HUVECs. Through quantitative transcriptional analysis, increased expression of vegfr2 (kdr, kdrl) and vegfr1 was detected after GXNT or SalB/FA treatment, together with upregulated transcription of their ligands including vegf-a, vegf-b, and pgfb. Bevacizumab, an anti-human VEGF-A monoclonal antibody, was able to significantly, but not completely, block the pro-angiogenic effects of GXNT or SalB/FA, suggesting their multi-targeting properties. CONCLUSIONS: In conclusion, from a traditional Chinese medicine with effects in enhancing blood circulation, we demonstrated the synergistic pro-angiogenic effects of Sal B and FA via both in vitro and in vivo models, which function at least partially through regulating the expression of VEGF receptors and ligands. Future studies are warranted to further elaborate the molecular interaction between these two compounds and the key regulators in the process of neovascularization.

Cytotoxic effects and tolerability of gemcitabine and axitinib in a xenograft model for c-myc amplified medulloblastoma.

Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.

NRP1 and MMP9 are dual targets of RNA-binding protein QKI5 to alter VEGF-R/ NRP1 signalling in trophoblasts in preeclampsia.

Preeclampsia (PE) is characterized by placental ischemia and hypoxia, resulting in abnormal casting of the uterine spiral artery, which is mainly caused by insufficient trophoblastic cell infiltration. A reduction in levels of growth factor-based signalling via Neuropilin-1 (NRP1) has been shown to contribute to dysfunctional trophoblast development. In this study, we showed that the RNA-binding protein, QKI5, regulated NRP1 expression and significantly improved trophoblast proliferation in vitro and in vivo. QKI5 and NRP1 expressions were significantly reduced in human PE placentas and in trophoblasts during hypoxia. Overexpression of these factors significantly improved cell proliferation and migration in vitro, in contrast to a decrease upon siRNA knockdown of QKI5 and NRP1 in HTR-8/SVneo cells. Using RIP and RNA pull-down assays, we further showed that QKI5 directly interacted with the 3'-UTR region of NRP1, to mediate cell proliferation and migration via matrix metalloprotease-9. Further, similar to NRP1, QKI5 also targets matrix metalloproteinase 9 (MMP9) involved in secretion of growth factors and its effects can be counteracted by NRP1 overexpression. In vivo studies using a PE mouse model revealed that QKI5 overexpression alleviated PE-related symptoms such as elevated blood pressure and proteinuria. Taken together, we found that QKI5 was a novel regulator, of VEGF-R/NRP1 signalling pathway functioning in trophoblast proliferation and migration, resulting in major contributors to the pathogenesis of PE. While careful evaluation of the broad implications of QKI5 expression is still necessary, this study identified QKI5 as a promising target for treatment strategies in acute PE patients.

Intermittent hypoxia changes the interaction of the kinin-VEGF system and impairs myocardial angiogenesis in the hypertrophic heart.

Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension-related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein-kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1-5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction.

New Therapeutic Opportunities for the Treatment of Squamous Cell Carcinomas: A Focus on Novel Driver Kinases.

Squamous cell carcinomas of the lung, head and neck, esophagus, and cervix account for more than two million cases of cancer per year worldwide with very few targetable therapies available and minimal clinical improvement in the past three decades. Although these carcinomas are differentiated anatomically, their genetic landscape shares numerous common genetic alterations. Amplification of the third chromosome's distal portion (3q) is a distinguishing genetic alteration in most of these carcinomas and leads to copy-number gain and amplification of numerous oncogenic proteins. This area of the chromosome harbors known oncogenes involved in squamous cell fate decisions and differentiation, including TP63, SOX2, ECT2, and PIK3CA. Furthermore, novel targetable oncogenic kinases within this amplicon include PRKCI, PAK2, MAP3K13, and TNIK. TCGA analysis of these genes identified amplification in more than 20% of clinical squamous cell carcinoma samples, correlating with a significant decrease in overall patient survival. Alteration of these genes frequently co-occurs and is dependent on 3q-chromosome amplification. The dependency of cancer cells on these amplified kinases provides a route toward personalized medicine in squamous cell carcinoma patients through development of small-molecules targeting these kinases.

Disabling VEGF-Response of Purkinje Cells by Downregulation of KDR via miRNA-204-5p.

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC.

VEGF-Independent Activation of Müller Cells by the Vitreous from Proliferative Diabetic Retinopathy Patients.

Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.

VER/VEGF receptors regulate AMPA receptor surface levels and glutamatergic behavior.

Several intracellular trafficking pathways contribute to the regulation of AMPA receptor (AMPAR) levels at synapses and the control of synaptic strength. While much has been learned about these intracellular trafficking pathways, a major challenge is to understand how extracellular factors, such as growth factors, neuropeptides and hormones, impinge on specific AMPAR trafficking pathways to alter synaptic function and behavior. Here, we identify the secreted ligand PVF-1 and its cognate VEGF receptor homologs, VER-1 and VER-4, as regulators of glutamate signaling in C. elegans. Loss of function mutations in ver-1, ver-4, or pvf-1, result in decreased cell surface levels of the AMPAR GLR-1 and defects in glutamatergic behavior. Rescue experiments indicate that PVF-1 is expressed and released from muscle, whereas the VERs function in GLR-1-expressing neurons to regulate surface levels of GLR-1 and glutamatergic behavior. Additionally, ver-4 is unable to rescue glutamatergic behavior in the absence of pvf-1, suggesting that VER function requires endogenous PVF-1. Inducible expression of a pvf-1 rescuing transgene suggests that PVF-1 can function in the mature nervous system to regulate GLR-1 signaling. Genetic double mutant analysis suggests that the VERs act together with the VPS-35/retromer recycling complex to promote cell surface levels of GLR-1. Our data support a genetic model whereby PVF-1/VER signaling acts with retromer to promote recycling and cell surface levels of GLR-1 to control behavior.

A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

Evaluation of Variances in VEGF-A-D and VEGFR-1-3 Expression in the Ishikawa Endometrial Cancer Cell Line Treated with Salinomycin and Anti-Angiogenic/Lymphangiogenic Effect.

BACKGROUND: In cancer, an excessive and uncontrolled process of creating new blood and lymphatic vessels that play a key role in the metastasis process can be observed. The Vascular Endothelial Growth Factor (VEGF-A,-B,-C,-D) family together with their specific receptors (VEGFR-1,-2,- 3) plays a key role in these processes, therefore, it would be reasonable to determine the correct pattern of their expression. OBJECTIVES: The study aimed to assess the use of salinomycin as an anti-angiogenic and anti-lymphangiogenic drug during endometrial cancer by examining changes in the expression pattern of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2 and VEGFR-3 depending on the treatment period of the Ishikawa endometrial cancer cells with salinomycin in comparison to the control culture. MATERIALS AND METHODS: To determine how influential salinomycin was on the expression of both mRNAs, 1 µM of the drug was added to the cell culture and then it was cultured all together for 12, 24 and 48 hour periods. The cells that made up the control culture were not treated with salinomycin. To determine the changes in the expression profile of the selected genes, we used the microarray, techniques: RTqPCR and ELISA (p<0.05). RESULTS: For all isoforms of VEGF-A-D as well as receptors of VEGFR-1-3, a decrease in expression under the influence of salinomycin was noted. For VEGF-A and VEGFR-1, the difference in the expression between the culture treated with salinomycin in comparison to the control was statistically significant (p=0.0004). In turn, for VEGF-B, the difference between the culture exposed for 24 hours in comparison to the control (p=0.00000) as well as the comparison between H48 vs. C (p=0.00000) was statistically significant. In reference to VEGF-C, VEGFR-2 and VEGFR-3, the statistical analysis showed the significant difference in expression between the culture incubated with the drug for 12, 24 and 48 hours in comparison to the control as well as between the selected times. For all of these comparisons, p=0.00000 was utilized. CONCLUSION: Salinomycin changes the expression pattern of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2, and VEGFR-3 in endometrial cancer cells. The obtained results suggest that salinomycin might exert the effect via VEGF signaling pathways.

Histone deacetylase 6 inhibition mitigates renal fibrosis by suppressing TGF-ß and EGFR signaling pathways in obstructive nephropathy.

We have recently shown that histone deacetylase 6 (HDAC6) is critically involved in the pathogenesis of acute kidney injury. Its role in renal fibrosis, however, remains unclear. In this study, we examined the effect of ricolinostat (ACY-1215), a selective inhibitor of HDAC6, on the development of renal fibrosis in a murine model induced by unilateral ureteral obstruction (UUO). HDAC6 was highly expressed in the kidney following UUO injury, which was coincident with deposition of collagen fibrils and expression of α-smooth muscle actin, fibronectin, and collagen type III. Administration of ACY-1215 reduced these fibrotic changes and inhibited UUO-induced expression of transforming growth factor-ß1 and phosphorylation of Smad3 while increasing expression of Smad7. ACY-1215 treatment also suppressed phosphorylation of epidermal growth factor receptor (EGFR) and several signaling molecules associated with renal fibrogenesis, including AKT, STAT3, and NF-κB in the injured kidney. Furthermore, ACY-1215 was effective in inhibiting dedifferentiation of renal fibroblasts to myofibroblasts and the fibrotic change of renal tubular epithelial cells in culture. Collectively, these results indicate that HDAC6 inhibition can attenuate development of renal fibrosis by suppression of transforming growth factor-ß1 and EGFR signaling and suggest that HDAC6 would be a potential therapeutic target for the treatment of renal fibrosis.

Inhibiting the NLRP3 inflammasome with MCC950 ameliorates retinal neovascularization and leakage by reversing the IL-1ß/IL-18 activation pattern in an oxygen-induced ischemic retinopathy mouse model.

Activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays an important role in ocular neovascularization. In our study, we found that the expression and activation levels of NLRP3 inflammasome components, including NLRP3, an apoptosis-associated speck-like protein (ASC) containing caspase activation and recruitment domain (CARD) and caspase-1 (CAS1), were significantly upregulated. In addition, we found interleukin (IL)-1ß activity increased while IL-18 activity decreased in the retinas of oxygen-induced ischemic retinopathy (OIR) mice. MCC950, an inhibitor of NLRP3, reversed the IL-1ß/IL-18 activation pattern, inhibited the formation of retinal neovascularization (RNV), decreased the number of acellular capillaries and reduced leakage of retinal vessels. Moreover, MCC950 could regulate the expression of endothelial cell- and pericyte function-associated molecules, such as vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1, VEGFR2, matrix metalloproteinase (MMP)2, MMP9, tissue inhibitor of metalloproteinases (TIMP)1, TIMP2, platelet-derived growth factor receptor-ß (PDGFR-ß), platelet-derived growth factor-B (PDGF-B), and angiopoietin2 (Ang2). In vitro, recombinant human (r)IL-18 and rIL-1ß regulated the expression of endothelial cell- and pericyte function-associated molecules and the proliferation and migration of endothelial cells and pericytes. We therefore determined that inhibiting the NLRP3 inflammasome with MCC950 can regulate the function of endothelial cells and pericytes by reversing the IL-1ß/IL-18 activation pattern to ameliorate RNV and leakage; thereby opening new avenues to treat RNV-associated ocular diseases.

Development of Human CBF1-Targeting Single-Stranded DNA Aptamers with Antiangiogenic Activity In Vitro.

C promoter binding factor 1 (CBF1) (alias RBPJ) is a critical transcription factor involved in Notch signaling. The activation of Notch signaling through CBF1 maintains the angiostatic state of endothelial cells suppressing angiogenesis, that is, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) induces angiogenesis by promoting the proteasomal degradation of CBF1, in addition to endothelial cell proliferation. To date, angiogenic inhibitors targeting VEGF have been successfully used in clinics for cancer and age-related macular degeneration. Most antiangiogenic drugs, however, only target VEGF or VEGF receptors. In this study, to expand the repertoire of antiangiogenic therapeutics, we developed 15 single-stranded deoxyribonucleic acid (ssDNA) aptamers capable of binding to CBF1 with high affinity (Kd; 10-300 nM). To this end, systematic evolution of ligands by the exponential enrichment (SELEX) method was applied. One of the CBF1-binding ssDNA aptamers, Apt-3, inhibited angiogenesis through the activation of Notch signaling in vitro. We found that Apt-3 directly interacted with the LAG1 domain of CBF1. We suggest that the Apt-3 ssDNA aptamer may contribute to the development of a novel angiogenic inhibitor, which does not target VEGF.

TRIM28 regulates sprouting angiogenesis through VEGFR-DLL4-Notch signaling circuit.

Sprouting angiogenesis is a highly coordinately process controlled by vascular endothelial growth factor receptor (VEGFR)-Notch signaling. Here we investigated whether Tripartite motif-containing 28 (TRIM28), which is an epigenetic modifier implicated in gene transcription and cell differentiation, is essential to mediate sprouting angiogenesis. We observed that knockdown of TRIM28 ortholog in zebrafish resulted in developmental vascular defect with disorganized and reduced vasculatures. Consistently, TRIM28 knockdown inhibited angiogenic sprouting of cultured endothelial cells (ECs), which exhibited increased mRNA levels of VEGFR1, Delta-like (DLL) 3, and Notch2 but reduced levels of VEGFR2, DLL1, DLL4, Notch1, Notch3, and Notch4.The regulative effects of TRIM28 on these angiogenic factors were partially mediated by hypoxia-inducible factor 1 α (HIF-1α) and recombination signal-binding protein for immunoglobulin kappa J region (RBPJκ). In vitro DNA-binding assay showed that TRIM28 knockdown increased the association of RBPJκ with DNA sequences containing HIF-1α-binding sites. Moreover, the phosphorylation of TRIM28 was controlled by VEGF and Notch1 through a mechanism involving RBPJκ-dual-specificity phosphatase (DUSP)-p38 MAPK, indicating a negative feedback mechanism. These findings established TRIM28 as a crucial regulator of VEGFR-Notch signaling circuit through HIF-1α and RBPJκ in EC sprouting angiogenesis.