Search

Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis. [ABSTRACT FROM AUTHOR]

Laryngeal cancer (LC) is the second most common head and neck cancer and has a decreasing 5-year survival rate worldwide. Circular RNAs (circRNAs) regulate cancer development in diverse ways based on their distinct biogenesis mechanisms and expansive regulatory roles. However, currently, there is little research on how exosomal circRNAs are involved in the development of LC. Here, we demonstrated that circPVT1, a circRNA derived from the well-studied long noncoding RNA PVT1, is correlated with disease progression in LC and promotes angiogenesis both in vivo and in vitro. Mechanistically, circPVT1 is loaded into LC cell-secreted exosomes and taken up by vascular epithelium cells. By sponging miR-30c-5p, exosomal circPVT1 promotes Rap1b expression, which dramatically enhances vascular endothelial growth factor receptor 2 and the phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation, ultimately resulting in the induction of angiogenesis. Furthermore, our xenograft models demonstrated that the combination of short hairpin RNA-circPVT1 and cetuximab showed high efficacy in inhibiting tumor growth and angiogenesis. Collectively, these findings uncover a novel mechanism of exosomal circRNA-mediated angiogenesis modulation and provide a preclinical rationale for testing this analogous combination in patients with LC. [ABSTRACT FROM AUTHOR]

Abstract Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis.

PURPOSE: This open-label, single-arm, phase II study evaluated the vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitor (TKI) rivoceranib in patients with recurrent or metastatic (R/M) adenoid cystic carcinoma (ACC). PATIENTS AND METHODS: Eligible patients had confirmed disease progression per Response Evaluation Criteria in Solid Tumors (RECIST) with ≥20% increase in radiologically or clinically measurable lesions or appearance of new lesions within the preceding 6 months. Patients received oral rivoceranib 700 mg once daily. Primary outcomes were objective response rate (ORR) by investigator review and by blinded independent review committee (BIRC). RESULTS: Eighty patients were enrolled and 72 were efficacy evaluable. Seventy-four patients had distant metastases and 49 received prior systemic treatment (14 received VEGFR TKIs). Per investigator and BIRC, respectively, ORR was 15.3% [95% confidence interval (95% CI), 7.9-25.7] and 9.7% (95% CI, 4.0-19.0); median duration of response was 14.9 months (95% CI, 4.9-17.3) and 7.2 months (95% CI, 3.5-8.4); and median progression-free survival was 9.0 months (95% CI, 7.3-11.5) and 9.0 months (95% CI, 7.7-11.5). Grade ≥3 treatment-related adverse events occurred in 56 patients (70.0%); the most common were hypertension (34, 42.5%) and stomatitis (6, 7.5%). Four grade 5 events occurred with one attributed to rivoceranib (epistaxis). Sixty-eight patients (85.0%) had ≥1 dose modifications and 16 patients (20.0%) discontinued rivoceranib for toxicity. CONCLUSIONS: In patients with progressing R/M ACC, rivoceranib demonstrated antitumor activity and a manageable safety profile consistent with other VEGFR TKIs.

New series of thiazolyl-pyrazoline derivatives (7a-7d, 10a-10d and 13a-13f) have been synthesised and assessed for their potential EGFR and VEGFR-2 inhibitory activities. Compounds 10b and 10d exerted potent and selective inhibitory activity towards the two receptor tyrosine kinases; EGFR (IC50 = 40.7 ± 1.0 and 32.5 ± 2.2 nM, respectively) and VEGFR-2 (IC50 = 78.4 ± 1.5 and 43.0 ± 2.4 nM, respectively). The best anti-proliferative activity for the examined thiazolyl-pyrazolines was observed against the non-small lung cancer cells (NSCLC). Compounds 10b and 10d displayed pronounced efficacy against A549 (IC50 = 4.2 and 2.9 µM, respectively) and H441 cell lines (IC50 = 4.8 and 3.8 µM, respectively). Moreover, our results indicated that 10b and 10d were much more effective towards EGFR-mutated NSCLC cell lines (NCI-H1650 and NCI-H1975 cells) than gefitinib. Finally, compounds 10b and 10d induce G2/M cell cycle arrest and apoptosis and inhibit migration in A549 cancerous cells.

Background: Rivoceranib is an oral, selective tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2. ANGEL (NCT03042611) was a global, randomized, double-blinded, placebo-controlled, phase 3 study evaluating rivoceranib as 3rd-line or ≥4th-line therapy in patients with advanced/metastatic gastric or gastroesophageal junction (GEJ) cancer. Methods: Patients had failed ≥2 lines of chemotherapy and were randomized 2:1 to rivoceranib 700 mg once daily or placebo with best supportive care. Primary endpoint: overall survival (OS) in the intention-to-treat population. Secondary endpoints: progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR) by blinded independent central review (BICR). Results: In total, 460 patients (rivoceranib n = 308, placebo n = 152) were enrolled. OS was not statistically different for rivoceranib versus placebo (median 5.78 vs. 5.13 months; hazard ratio [HR] 0.93, 95% CI 0.74–1.15; p = 0.4724). PFS by BICR (median 2.83 vs. 1.77 months; HR 0.58, 95% CI 0.47–0.71; p < 0.0001), ORR (6.5% vs. 1.3%; p = 0.0119), and DCR (40.3 vs. 13.2%; p < 0.0001) were improved with rivoceranib versus placebo. In patients receiving ≥4th-line therapy, OS (median 6.34 vs. 4.73 months; p = 0.0192) and PFS by BICR (median 3.52 vs. 1.71 months; p < 0.0001) were improved with rivoceranib versus placebo. The most common grade ≥ 3 treatment-emergent adverse events with rivoceranib were hypertension (17.9%), anemia (10.4%), aspartate aminotransferase increased (9.4%), asthenia (8.5%), and proteinuria (7.5%). Conclusions: This study did not meet its primary OS endpoint. Compared to placebo, rivoceranib improved PFS, ORR, and DCR. Rivoceranib also improved OS in a prespecified patient subgroup receiving ≥4th-line therapy. [ABSTRACT FROM AUTHOR]

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

BackgroundPreclinical data suggest that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β signaling interact to stimulate angiogenesis and suppress antitumor immune responses. Thus, combined inhibition of both pathways may offer greater antitumor activity compared with VEGF-targeted antiangiogenic monotherapy against hepatocellular carcinoma (HCC).MethodsThis is a multicenter, open-label, phase 1b study of galunisertib, an inhibitor of TGF-β receptor 1, and ramucirumab, an anti-VEGF receptor 2 antibody, in patients with advanced HCC aiming to define the maximum tolerated dose (MTD). Secondary objectives included safety, pharmacokinetics (PK), antitumor efficacy, and plasma alpha-fetoprotein and TGF-β kinetics. Dose escalation employed a 3 + 3 design. Patients received galunisertib at 80 mg (cohort 1) or 150 mg (cohort 2) orally twice a day on days 1-14 of a 28-day cycle combined with ramucirumab 8 mg/kg intravenously every 2 weeks.ResultsEight patients were enrolled: three in cohort 1 and five in cohort 2 (two patients were unevaluable due to rapid disease progression and replaced). No dose-limiting toxicities were observed. Treatment-related adverse events (AEs) of any grade in ≥2 patients included nausea (25%) and vomiting (25%). There was one Grade 3 treatment-related AE, a cerebrovascular accident possibly related to ramucirumab. Galunisertib exposure was dose-proportional and not affected by ramucirumab. The RECIST version 1.1 objective response rate and disease control rate were 0% and 12.5%, respectively.ConclusionCombination therapy was safe and tolerable and displayed favorable PK. The MTD was established at galunisertib at 150 mg orally twice a day and ramucirumab 8 mg/kg intravenously every 2 weeks. The results do not support the preclinical hypothesis that blocking TGFβ signaling enhances efficacy of VEGF-targeted therapy; thus further clinical development was halted for the combination of galunisertib and ramucirumab.

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.

Slug (SNAI2), a member of the well-conserved Snail family of transcription factors, has multiple developmental roles, including in epithelial-to-mesenchymal transition (EMT). Here, we show that Slug is critical for the pathological angiogenesis needed to sustain tumor growth, and transiently necessary for normal developmental angiogenesis. We find that Slug upregulation in angiogenic endothelial cells (EC) regulates an EMT-like suite of target genes, and suppresses Dll4-Notch signaling thereby promoting VEGFR2 expression. Both EC-specific Slug re-expression and reduced Notch signaling, either by γ-secretase inhibition or loss of Dll4, rescue retinal angiogenesis in SlugKO mice. Conversely, inhibition of VEGF signaling prevents excessive angiogenic sprouting of Slug overexpressing EC. Finally, endothelial Slug (but not Snail) is activated by the pro-angiogenic factor SDF1α via its canonical receptor CXCR4 and the MAP kinase ERK5. Altogether, our data support a critical role for Slug in determining the angiogenic response during development and disease.

Human endothelial cells (ECs) are important tools in research and development of new therapies in the fields of angiogenesis, vasculogenesis, engineering organoids and multicellular tissues, drug discovery, and disease modeling. Efficient and robust induction of ECs from human pluripotent stem cells (hPSCs) serve as a renewable and indefinite cell sources. However, individual lines of embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are distinct and can often respond very differently to the same microenvironmental cues. Therefore, we set out to develop a differentiation methodology specifically designed for robustness across multiple human iPSC lines. In general, the key soluble signals remain consistent across cell lines, but because the differentiation and proliferation kinetics can differ slightly in hESC and iPSC cell lines, the time point for KDR+ cell sorting must be pre-determined for each cell line. This three-stage induction method uses three different chemically defined medium formulations and generates highly purified populations of actively proliferating and functional VE-cadherin+ ECs within 30 days.

PurposeVascular endothelial growth factor receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1) are two prominent synergistic receptors overexpressed on new blood vessels in glioma and may be promising targets for antiglioma therapy. The aim of this study was to design a dual receptor targeting and blood-brain barrier (BBB) penetrating peptide-modified polyethyleneimine (PEI) nanocomplex that can efficiently deliver the angiogenesis-inhibiting secretory endostatin gene (pVAXI-En) to treat glioma.Materials and methodsWe first constructed the tandem peptide TAT-AT7 by conjugating AT7 to TAT and evaluated its binding affinity to VEGFR-2 and NRP-1, vasculature-targeting ability and BBB crossing capacity. Then, TAT-AT7-modified PEI polymer (PPTA) was synthesized, and a pVAXI-En-loaded PPTA nanocomplex (PPTA/pVAXI-En) was prepared. The physicochemical properties, cytotoxicity, transfection efficiency, capacities to cross the BBB and BTB (blood-tumor barrier) and glioma-targeting properties of PPTA/pVAXI-En were investigated. Moreover, the in vivo anti-angiogenic behaviors and anti-glioma effects of PPTA/pVAXI-En were evaluated in nude mice.ResultsThe binding affinity of TAT-AT7 to VEGFR-2 and NRP-1 was approximately 3 to 10 times greater than that of AT7 or TAT. The cellular uptake of TAT-AT7 in endothelial cells was 5-fold and 119-fold greater than that of TAT and AT7 alone, respectively. TAT-AT7 also displayed remarkable efficiency in penetrating the BBB and glioma tissue in vivo. PPTA/pVAXI-En exhibited lower cytotoxicity, stronger BBB and BTB traversing abilities, higher selective glioma targeting and better gene transfection efficiency than PEI/pVAXI-En. More importantly, PPTA/pVAXI-En significantly suppressed the tube formation and migration of endothelial cells, inhibited glioma growth, and reduced the microvasculature in orthotopic U87 glioma-bearing nude mice.ConclusionOur study demonstrates that PPTA/pVAXI-En can be exploited as an efficient dual-targeting nanocomplex to cross the BBB and BTB, and hence it represents a feasible and promising nonviral gene delivery system for effective glioma therapy.

PurposeAmplifications of receptor tyrosine kinases (RTKS) are therapeutic targets in multiple tumor types (e.g. HER2 in breast cancer), and amplification of the chromosome 4 segment harboring the three RTKs KIT, PDGFRA, and KDR (4q12amp) may be similarly targetable. The presence of 4q12amp has been sporadically reported in small tumor specific series but a large-scale analysis is lacking. We assess the pan-cancer landscape of 4q12amp and provide early clinical support for the feasibility of targeting this amplicon.Experimental designTumor specimens from 132,872 patients with advanced cancer were assayed with hybrid capture based comprehensive genomic profiling which assays 186-315 genes for all classes of genomic alterations, including amplifications. Baseline demographic data were abstracted, and presence of 4q12amp was defined as 6 or more copies of KIT/KDR/PDGFRA. Concurrent alterations and treatment outcomes with matched therapies were explored in a subset of cases.ResultsOverall 0.65% of cases harbored 4q12amp at a median copy number of 10 (range 6-344). Among cancers with >100 cases in this series, glioblastomas, angiosarcomas, and osteosarcomas were enriched for 4q12amp at 4.7%, 4.8%, and 6.4%, respectively (all p < 0.001), giving an overall sarcoma (n = 6,885) incidence of 1.9%. Among 99 pulmonary adenocarcinoma cases harboring 4q12amp, 50 (50%) lacked any other known driver of NSLCC. Four index cases plus a previously reported case on treatment with empirical TKIs monotherapy had stable disease on average exceeding 20 months.ConclusionWe define 4q12amp as a significant event across the pan-cancer landscape, comparable to known pan-cancer targets such as NTRK and microsatellite instability, with notable enrichment in several cancers such as osteosarcoma where standard treatment is limited. The responses to available TKIs observed in index cases strongly suggest 4q12amp is a druggable oncogenic target across cancers that warrants a focused drug development strategy.Implications for practiceCoamplification of the receptor tyrosine kinases (rtks) KIT/KDR/PDGFRA (4q12amp) is present broadly across cancers (0.65%), with enrichment in osteosarcoma and gliomas. Evidence for this amplicon having an oncogenic role is the mutual exclusivity of 4q12amp to other known drivers in 50% of pulmonary adenocarcinoma cases. Furthermore, preliminary clinical evidence for driver status comes from four index cases of patients empirically treated with commercially available tyrosine kinase inhibitors with activity against KIT/KDR/PDGFRA who had stable disease for 20 months on average. The sum of these lines of evidence suggests further clinical and preclinical investigation of 4q12amp is warranted as the possible basis for a pan-cancer drug development strategy.

Simple Summary: Lung cancer is the most common and lethal malignancy worldwide. Musashi-2 (MSI2) is an RNA-binding protein that is overexpressed in advanced NSCLC. VEGFR2 protein expression contributes to NSCLC progression and several FDA-approved drugs are used to target it in the clinic. Here, we show that MSI2 is a strong positive regulator of VEGFR2 protein levels in murine and human NSCLC cell lines. Furthermore, we found that MSI2 protein directly binds to VEGFR2 and PTEN mRNAs and impacts VEGFR2 downstream signaling, in part via PTEN regulation. Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESC) are excellent cell culture systems for elucidating developmental signals that may be part of the stem cell niche. Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche can also play a role in directing cells towards differential cell lineages. Interested in diverging vascular fates, we set out to examine to what extent mechanical signaling played a role in endothelial cell and/or smooth muscle fate. Using chemically-defined staged vascular differentiation methods, vascular progenitor cells (VPC) fate was examined on single stiffness polyacrylamide hydrogels of 10 kPa, 40 kPa and >0.1 GPa. Emergence of vascular cell populations aligned with corresponding hydrogel stiffness: EC-lineages favoring the softer material and SMC lineages favoring the stiffest material. Statistical significance was observed on both cell lines on almost all days. Transcriptome analysis indicated that the populations on the varying stiffness emerge in distinct categories. Lastly, blocking studies show that αvβ1, and not αvβ6, activation mediates stiffness-directed vascular differentiation. Overall, these studies indicate that softer materials direct VPCs into a more EC-like fate compared to stiffer materials. STATEMENT OF SIGNIFICANCE: Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche also plays a role in directing cell fate. Several studies have examined the stiffness-induced cell fate from mesenchymal stem cells (MSCs) and undifferentiated embryonic stem cells (ESCs). This is the first study that rigorously examines the role of matrix stiffness in diverging vascular fates from a purified population of vascular progenitor cells (VPCs). We show that the emergence of endothelial cell (EC) versus smooth muscle cell (SMC) populations corresponds with hydrogel stiffness: EC-lineages favoring the softness material and SMC lineages favoring the stiffest material, and that αvβ1 activation mediates this stiffness-directed vascular differentiation.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands.

Pial collaterals provide protection from ischemic damage and improve the prognosis of stroke patients. The origin or precise sequence of events underlying pial collateral development is unclear and has prevented clinicians from adapting new vascularization and regeneration therapies. We use genetic lineage tracing and intravital imaging of mouse brains at cellular resolution to show that during embryogenesis, pial collateral arteries develop from extension and anastomoses of pre-existing artery tips in a VegfR2-dependent manner. This process of artery tip extension occurs on pre-determined microvascular tracks. Our data demonstrate that an arterial receptor, Cxcr4, earlier shown to drive artery cell migration and coronary collateral development, is dispensable for the formation and maintenance of pial collateral arteries. Our study shows that collateral arteries of the brain are built by a mechanism distinct from that of the heart. [Display omitted] • Pial collaterals originate from VegfR2-dependent cerebral artery tip extensions • Pial artery tip extension is preceded by microvascular guidance • Cellular distribution of adult pial collaterals does not change, even without arterial VegfR2 • Arterial Cxcr4 is dispensable for pial collateral formation and its maintenance Pial collaterals in mice form by VegfR2-dependent artery tip extensions along pre-determined microvascular tracks. This mechanism is independent of arterial Cxcr4, a vascular receptor key to coronary collateral development in mice. Kumar et al. provide support for two distinct organ-specific mechanisms that build collateral arteries in the heart and brain. [ABSTRACT FROM AUTHOR]

RationalePulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females.ObjectiveTo evaluate whether and how Treg deficiency differentially affects male and female rats in experimental PH.Methods and resultsMale and female athymic rnu/rnu rats, lacking Tregs, were treated with the VEGFR2 (vascular endothelial growth factor receptor 2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution before SU5416 administration. Plasma PGI2 (prostacyclin) levels were measured. Lung and right ventricles were assessed for the expression of the vasoprotective proteins COX-2 (cyclooxygenase 2), PTGIS (prostacyclin synthase), PDL-1 (programmed death ligand 1), and HO-1 (heme oxygenase 1). Inhibitors of these pathways were administered to athymic rats undergoing Treg immune reconstitution. Finally, human cardiac microvascular endothelial cells cocultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and ER (estrogen receptor) expression, and culture supernatants were assayed for PGI2 and IL (interleukin)-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented right ventricular fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1, and PDL-1 expression and reduced right ventricular PDL-1 levels. In both sexes, Treg immune reconstitution protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and PD-1 (programmed death 1)/PDL-1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL-1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10.ConclusionsIn 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

Angiogenesis inhibitor drugs targeting vascular endothelial growth factor (VEGF) signaling to the endothelial cell (EC) are used to treat various cancer types. However, primary or secondary resistance to therapy is common. Clinical and pre‐clinical studies suggest that alternative pro‐angiogenic factors are upregulated after VEGF pathway inhibition. Therefore, identification of alternative pro‐angiogenic pathway(s) is critical for the development of more effective anti‐angiogenic therapy. Here we study the role of apelin as a pro‐angiogenic G‐protein‐coupled receptor ligand in tumor growth and angiogenesis. We found that loss of apelin in mice delayed the primary tumor growth of Lewis lung carcinoma 1 and B16F10 melanoma when combined with the VEGF receptor tyrosine kinase inhibitor, sunitinib. Targeting apelin in combination with sunitinib markedly reduced the tumor vessel density, and decreased microvessel remodeling. Apelin loss reduced angiogenic sprouting and tip cell marker gene expression in comparison to the sunitinib‐alone‐treated mice. Single‐cell RNA sequencing of tumor EC demonstrated that the loss of apelin prevented EC tip cell differentiation. Thus, apelin is a potent pro‐angiogenic cue that supports initiation of tumor neovascularization. Together, our data suggest that targeting apelin may be useful as adjuvant therapy in combination with VEGF signaling inhibition to inhibit the growth of advanced tumors. [ABSTRACT FROM AUTHOR]

Background: BC001 is a novel fully human immunoglobulin G1 monoclonal antibody blocking VEGFR2. This phase I study aimed to assess BC001 alone and plus chemotherapy in solid tumors., Methods: In dose escalation part, BC001 was assessed at dose levels of 2, 4, 8, 12, 16 mg/kg on day 1,15, every 28 days, followed an accelerated titration and "3 + 3" design. BC001 plus paclitaxel was assessed at dose level of 8, 10 mg/kg. The primary endpoints included the DLT, MTD and RDE of BC001. In dose expansion part, it aimed to assess the anti-tumor activity of BC001 alone and plus chemotherapy in gastric cancer as 2nd line treatment., Results: Overall, 53 patients were finally enrolled in this study (phase Ia, n = 28; phase Ib, n = 25). In phase Ia part, 1 DLT (grade 4 neutropenia lasting 4 days) was observed in BC001(8 mg/kg) plus paclitaxel cohort. MTD was not reached. All patients experienced TEAEs of any grade. Twenty-four of them suffered ≥ grade 3 TEAEs. leukopenia (n = 33, 62.3%), hemoglobin decreased (n = 32, 60.4%), neutropenia (n = 29, 54.7%) were commonly observed hematological toxicities. The half-life of 140-240 h. And the PK parameters were not largely influenced by combination with paclitaxel. The serum sVEGFR-1, VEGF-A, sVEGFR-2 could not predict the efficacy. Based on the safety, PK and efficacy data, BC001 of 8 mg/kg was determined as RED. Among the GC patients(n = 21) who receiving BC001 plus paclitaxel as 2nd-line treatment in phase 1b part, 6 patients achieved PR and 10 patients experienced SD. The ORR was 28.6% (95% CI 11.3%, 52.2%) and the DCR was 76.2% (95% CI 52.8%, 91.8%), with the median PFS of 5.4 months (95% CI 1.9, 7.0) and OS of 9.4 months (95% CI 5.4, NA). The median DoR was 5.1 months (95% CI 2.6, NA)., Conclusions: BC001 showed acceptable safety profile and preliminary response in both single-agent and combination with chemotherapy cohorts, especially in GC., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine-metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro-angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation-specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK-deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation-induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.

Hippocampal dentate gyrus is a focus of enhanced neurogenesis and excitability after traumatic brain injury. Increased neurogenesis has been proposed to aid repair of the injured network. Our data show that an early increase in neurogenesis after fluid percussion concussive brain injury is transient and is followed by a persistent decrease compared with age-matched controls. Post-injury changes in neurogenesis paralleled changes in neural precursor cell proliferation and resulted in a long-term decline in neurogenic capacity. Targeted pharmacology to restore post-injury neurogenesis to control levels reversed the long-term decline in neurogenic capacity. Limiting post-injury neurogenesis reduced early increases in dentate excitability and seizure susceptibility. Our results challenge the assumption that increased neurogenesis after brain injury is beneficial and show that early post-traumatic increases in neurogenesis adversely affect long-term outcomes by exhausting neurogenic potential and enhancing epileptogenesis. Treatments aimed at limiting excessive neurogenesis can potentially restore neuroproliferative capacity and limit epilepsy after brain injury.

Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.

Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes.

IntroductionThe physiologic vitamin D (VD), 1α,25(OH)2D3 (1,25D) is a local paracrine/autocrine effecter of fetal lung maturation. By stimulating alveolar type II cell and lipofibroblast proliferation and differentiation, parenterally administered 1,25D has been shown to enhance neonatal lung maturation; but due to the potential systemic side effects of the parenteral route, the translational value of these findings might be limited. To minimize the possibility of systemic toxicity, we examined the effects of VD on neonatal lung maturation, when delivered directly to lungs via nebulization.MethodsOne-day-old rat pups were administered three different doses of 1,25D and its physiologic precursor 25(OH)D (25D), or the diluent, via nebulization daily for 14 days. Pups were sacrificed for lung, kidneys, and blood collection to determine markers of lung maturation, and serum 25D and calcium levels.ResultsCompared to controls, nebulized 25D and 1,25D enhanced lung maturation as evidenced by the increased expression of markers of alveolar epithelial (SP-B, leptin receptor), mesenchymal (PPARγ, C/EBPα), and endothelial (VEGF, FLK-1) differentiation, surfactant phospholipid synthesis, and lung morphology without any significant increases in serum 25D and calcium levels.ConclusionsInhaled VD is a potentially safe and effective novel strategy to enhance neonatal lung maturation.

Canine transitional cell carcinoma (cTCC) is the most common naturally occurring bladder cancer and accounts for 1-2% of canine tumors. The prognosis is poor due to the high rate of invasiveness and metastasis at diagnosis. Sorafenib is a multi-kinase inhibitor that targets rapidly accelerated fibrosarcoma (RAF), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor-β (PDGFR-β), and KIT. In previous studies, a somatic mutation of B-rapidly accelerated fibrosarcoma (BRAF) and expressions of VEGFR-2 and PDGFR-β were observed in over 80% of patients with cTCC. Therefore, in this study, we investigated the anti-tumor effects of sorafenib on cTCC. Five cTCC cell lines were used in the in vitro experiments. All five cTCC cell lines expressed VEGFR-2 and PDGFR-β and sorafenib showed growth inhibitory effect on cTCC cell lines. Cell cycle arrest at the G0/G1 phase and subsequent apoptosis were observed following sorafenib treatment. In the in vivo experiments, cTCC (Sora) cells were subcutaneously injected into nude mice. Mice were orally administered with sorafenib (30 mg/kg daily) for 14 days. Sorafenib inhibited tumor growth compared to vehicle control. The necrotic area in the tumor tissues was increased in the sorafenib-treated group. Sorafenib also inhibited angiogenesis in the tumor microenvironment. Thus, sorafenib may be potential therapeutic agent for cTCC via its direct anti-tumor effect and inhibition of angiogenesis. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Orofacial Orthopedics/Fortschritte der Kieferorthopadie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Introduction: Ramucirumab (RAM) plus erlotinib was found to have superior progression-free survival (PFS) versus placebo plus erlotinib in untreated EGFR-mutated metastatic NSCLC in the global phase 3 RELAY study. RELAY+ was an open-label, two-period, single-arm, exploratory study of RAM plus gefitinib (GEF; period 1) and RAM plus osimertinib (period 2) in East Asia (NCT02411448). Methods: Period 1 evaluated RAM (10 mg/kg) plus GEF (250 mg/d) in patients with untreated EGFR-mutated metastatic NSCLC. Period 2 evaluated RAM plus osimertinib (80 mg/d) in patients with disease progression who acquired T790M mutation in period 1. Exploratory end points included 1-year PFS rate (primary), other efficacy parameters, safety, and biomarker analyses of plasma (baseline, on-treatment, follow-up) using next-generation sequencing. Results: From December 2017 to August 2018, a total of 82 patients were enrolled and started treatment (period 1, RAM + GEF). The 1-year PFS rate was 62.9% (95% confidence interval: 50.3–73.1). Treatment-emergent adverse events of grade three or higher were reported with RAM plus GEF in 60 of 82 patients (73.2%; five patients [6.1%] grade four). There were two deaths owing to adverse events that occurred (acute cardiac failure, congestive cardiac failure). T790M rate at disease progression in plasma was 81.0% (13 of 16 patients). Conclusions: RELAY+ was found to have a favorable benefit–risk profile for RAM plus GEF in first-line treatment of East Asian patients with EGFR-mutated NSCLC.

The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.

BackgroundIn glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression.MethodsMining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence.ResultsIn human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated β-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced.ConclusionsVEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells.

The present study sought to determine the anti-tumor effects of OXA-11, a potent, novel small-molecule amino pyrimidine inhibitor (1.2 pM biochemical IC(50)) of focal adhesion kinase (FAK). In studies of cancer cell lines, OXA-11 inhibited FAK phosphorylation at phospho-tyrosine 397 with a mechanistic IC(50) of 1 nM in TOV21G tumor cells, which translated into functional suppression of proliferation in 3-dimensional culture with an EC(50) of 9 nM. Studies of OXA-11 activity in TOV21G tumor-cell xenografts in mice revealed a pharmacodynamic EC(50) of 1.8 nM, indicative of mechanistic inhibition of pFAK [Y397] in these tumors. OXA-11 inhibited TOV21G tumor growth in a dose-dependent manner and also potentiated effects of cisplatin on tumor cell proliferation and apoptosis in vitro and on tumor growth in mice. Studies of pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice revealed OXA-11 suppression of pFAK [Y397] and pFAK [Y861] in tumors and liver. OXA-11 given daily from age 14 to 17 weeks reduced tumor vascularity, invasion, and when given together with the anti-VEGFR-2 antibody DC101 reduced the incidence, abundance, and size of liver metastases. Liver micrometastases were found in 100 % of mice treated with vehicle, 84 % of mice treated with OXA-11, and 79 % of mice treated with DC101 (19-24 mice per group). In contrast, liver micrometastases were found in only 52 % of 21 mice treated with OXA-11 plus DC101, and those present were significantly smaller and less numerous. Together, these findings indicate that OXA-11 is a potent and selective inhibitor of FAK phosphorylation in vitro and in vivo. OXA-11 slows tumor growth, potentiates the anti-tumor actions of cisplatin and--when combined with VEGFR-2 blockade--reduces metastasis of pancreatic neuroendocrine tumors in RIP-Tag2 mice.

Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium-targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin-interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium-specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Introduction: Intrahepatic cholangiocarcinoma (ICC), which is difficult to diagnose and is usually fatal due to its late clinical presentation and a lack of effective treatment, has risen over the past decades but without much improvement in prognosis. Objective: The study aimed to investigate the role of apatinib that targets vascular endothelial growth factor receptor-2 (VEGFR2) in ICC. Methods: MTT assays, cell scratch assays, and tube formation assays were used to assess the effect of apatinib on human ICC cell line (HuCCT-1) and RBE cells proliferation, migration, and angiogenic capacity, respectively. Expression of vascular endothelial growth factor (VEGF), VEGFR2, signal transducer and activator of transcription factor 3 (STAT3), pSTAT3, and hypoxia inducible factor 1 subunit alpha (HIF-1α) pathway proteins was assessed using Western blotting and mRNA expression analysis in HuCCT-1 was performed using RT-qPCR assays. The pcDNA 3.1(-)-VEGFR2 and pcDNA 3.1(-)-HIF-1α were transfected into HuCCT-1 and RBE cells using Lipofectamine 2,000 to obtain overexpressed HuCCT-1 and RBE cells. Results: We found that apatinib-inhibited proliferation, migration, and angiogenesis of HuCCT-1 and RBE cells in vitro in a dose-dependent manner. We also proved that apatinib effectively inhibits angiogenesis in tumor cells by blocking the expression of VEGF and VEGFR2 in these cells. In addition, we demonstrated that apatinib regulates the expression of STAT3 phosphorylation by inhibiting VEGFR2. Finally, we showed that apatinib regulates ICC angiogenesis and HIF-1α/VEGF expression via STAT3. Conclusions: Based on the above findings, we conclude that apatinib inhibits HuCCT-1 and RBE cell proliferation, migration, and tumor angiogenesis by inhibiting the VEGFR2/STAT3/HIF-1α axis signaling pathway. Apatinib can be a promising drug for ICC-targeted molecular therapy. [ABSTRACT FROM AUTHOR]

PurposeFit-for-purpose pharmacodynamic biomarkers could expedite development of combination antiangiogenic regimens. Plasma sVEGFR2 concentrations ([sVEGFR2]) mark sunitinib effects on the systemic vasculature. We hypothesized that cilengitide would impair microvasculature recovery during sunitinib withdrawal and could be detected through changes in [sVEGFR2].Experimental designAdvanced solid tumor patients received 50 mg sunitinib daily for 14 days. For the next 14 days, patients were randomized to arm A (cilengitide 2,000 mg administered intravenously twice weekly) or arm B (no treatment). The primary endpoint was change in [sVEGFR2] between days 14 and 28. A candidate pharmacodynamic biomarker of cilengitide inhibition of integrin αvβ3, serum c-telopeptide collagen crosslinks (CTx), was also measured.ResultsOf 21 patients, 14 (7 per arm) received all treatments without interruption and had all blood samples available for analysis. The mean change and SD of [sVEGFR2] for all sunitinib-treated patients was consistent with previous data. There was no significant difference in the mean change in [sVEGFR2] from days 14 to 28 between the arms [arm A: 2.8 ng/mL; 95% confidence interval (CI), 2.1-3.6 vs. arm B: 2.0 ng/mL; 95% CI, 0.72-3.4; P = 0.22, 2-sample t test]. Additional analyses suggested (i) prior bevacizumab therapy to be associated with unusually low baseline [sVEGFR2] and (ii) sunitinib causes measurable changes in CTx.ConclusionsCilengitide had no measurable effects on any circulating biomarkers. Sunitinib caused measurable declines in serum CTx. The properties of [sVEGFR2] and CTx observed in this study inform the design of future combination antiangiogenic therapy trials.

Antiangiogenic therapy is commonly used for the treatment of colorectal cancer (CRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. The MET signaling pathway has been proposed to be a major contributor of resistance to antiangiogenic therapy. MET is upregulated in response to vascular endothelial growth factor pathway inhibition and plays an essential role in tumorigenesis and progression of tumors. In this study, we set out to determine the efficacy of cabozantinib in a preclinical CRC patient-derived tumor xenograft model. We demonstrate potent inhibitory effects on tumor growth in 80% of tumors treated. The greatest antitumor effects were observed in tumors that possess a mutation in the PIK3CA gene. The underlying antitumor mechanisms of cabozantinib consisted of inhibition of angiogenesis and Akt activation and significantly decreased expression of genes involved in the PI3K pathway. These findings support further evaluation of cabozantinib in patients with CRC. PIK3CA mutation as a predictive biomarker of sensitivity is intriguing and warrants further elucidation. A clinical trial of cabozantinib in refractory metastatic CRC is being activated.

Treatment of glioblastoma (GBM), the most common primary malignant brain tumor in adults, remains a significant unmet need in oncology. Historically, cytotoxic treatments provided little durable benefit, and tumors recurred within several months. This has spurred a substantial research effort to establish more effective therapies for both newly diagnosed and recurrent GBM. In this context, antiangiogenic therapy emerged as a promising treatment strategy because GBMs are highly vascular tumors. In particular, GBMs overexpress vascular endothelial growth factor (VEGF), a proangiogenic cytokine. Indeed, many studies have demonstrated promising radiographic response rates, delayed tumor progression, and a relatively safe profile for anti-VEGF agents. However, randomized phase III trials conducted to date have failed to show an overall survival benefit for antiangiogenic agents alone or in combination with chemoradiotherapy. These results indicate that antiangiogenic agents may not be beneficial in unselected populations of patients with GBM. Unfortunately, biomarker development has lagged behind in the process of drug development, and no validated biomarker exists for patient stratification. However, hypothesis-generating data from phase II trials that reveal an association between increased perfusion and/or oxygenation (ie, consequences of vascular normalization) and survival suggest that early imaging biomarkers could help identify the subset of patients who most likely will benefit from anti-VEGF agents. In this article, we discuss the lessons learned from the trials conducted to date and how we could potentially use recent advances in GBM biology and imaging to improve outcomes of patients with GBM who receive antiangiogenic therapy.

Abnormal blood rheological properties seldom occur in isolation and instead are accompanied by other complications, often designated as co-morbidities. In the metabolic syndrome with complications like hypertension, diabetes and lack of normal microvascular blood flow, the underlying molecular mechanisms that simultaneously lead to elevated blood pressure and diabetes as well as abnormal microvascular rheology and other cell dysfunctions have remained largely unknown. In this review, we propose a new hypothesis for the origin of abnormal cell functions as well as multiple co-morbidities. Utilizing experimental models for the metabolic disease with diverse co-morbidities we summarize evidence for the presence of an uncontrolled extracellular proteolytic activity that causes ectodomain receptor cleavage and loss of their associated cell function. We summarize evidence for unchecked degrading proteinase activity, e.g. due to matrix metalloproteases, in patients with hypertension, Type II diabetes and obesity, in addition to evidence for receptor cleavage in the form of receptor fragments and decreased extracellular membrane expression levels. The evidence suggest that a shift in blood rheological properties and other co-morbidities may in fact be derived from a common mechanism that is due to uncontrolled proteolytic activity, i.e. an early form of autodigestion. Identification of the particular proteases involved and the mechanisms of their activation may open the door to treatment that simultaneously targets multiple co-morbidities in the metabolic syndrome.

PurposeWe hypothesized that bortezomib, an agent that suppresses HIF-1α transcriptional activity, when combined with bevacizumab, would obviate the HIF-1α resistance pathway. The objectives of this phase I trial were to assess safety and biological activity of this combination.Experimental designPatients with advanced, refractory malignancies were eligible. Patients received bevacizumab and bortezomib (3-week cycle) with dose expansions permitted if responses were seen and for assessing correlates. Pharmacodynamic assessment included plasma VEGF, VEGFR2, 20S proteasome inhibition, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and HIF-1α tumor expression.ResultsNinety-one patients were treated (median=6 prior treatments). The FDA-approved doses of both drugs were safely reached, and the recommended phase 2 dose (RP2D) is bevacizumab 15 mg/kg with bortezomib 1.3 mg/m(2). Four patients attained partial response (PR) and seven patients achieved stable disease (SD) ≥ 6 months (Total SD ≥ 6 months/PR=11 (12%)). The most common drug-related toxicities included thrombocytopenia (23%) and fatigue (19%). DCE-MRI analysis demonstrated no dose-dependent decreases in K(trans) although analysis was limited by small sample size (N=12).ConclusionCombination bevacizumab and bortezomib is well-tolerated and has demonstrated clinical activity in patients with previously treated advanced malignancy. Pharmacodynamic assessment suggests that inhibition of angiogenic activity was achieved.

Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1-enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C-producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.

RationaleLymphatic vessels in the respiratory tract normally mature into a functional network during the neonatal period, but under some pathological conditions they can grow as enlarged, dilated sacs that result in the potentially lethal condition of pulmonary lymphangiectasia.ObjectiveWe sought to determine whether overexpression of the lymphangiogenic growth factor (vascular endothelial growth factor-C [VEGF-C]) can promote lymphatic growth and maturation in the respiratory tract. Unexpectedly, perinatal overexpression of VEGF-C in the respiratory epithelium led to a condition resembling human pulmonary lymphangiectasia, a life-threatening disorder of the newborn characterized by respiratory distress and the presence of widely dilated lymphatics.Methods and resultsAdministration of doxycycline to Clara cell secretory protein-reverse tetracycline-controlled transactivator/tetracycline operator-VEGF-C double-transgenic mice during a critical period from embryonic day 15.5 to postnatal day 14 was accompanied by respiratory distress, chylothorax, pulmonary lymphangiectasia, and high mortality. Enlarged sac-like lymphatics were abundant near major airways, pulmonary vessels, and visceral pleura. Side-by-side comparison revealed morphological features similar to pulmonary lymphangiectasia in humans. The condition was milder in mice given doxycycline after age postnatal day 14 and did not develop after postnatal day 35. Mechanistic studies revealed that VEGF recptor (VEGFR)-3 alone drove lymphatic growth in adult mice, but both VEGFR-2 and VEGFR-3 were required for the development of lymphangiectasia in neonates. VEGFR-2/VEGFR-3 heterodimers were more abundant in the dilated lymphatics, consistent with the involvement of both receptors. Despite the dependence of lymphangiectasia on VEGFR-2 and VEGFR-3, the condition was not reversed by blocking both receptors together or by withdrawing VEGF-C.ConclusionsThe findings indicate that VEGF-C overexpression can induce pulmonary lymphangiectasia during a critical period in perinatal development.