Explore the vast range of titles available.

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3 , but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2 +/− ; Vegfr3 +/− compound heterozygosity recapitulated homozygous loss of Vegfr3 . These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts. Notch and VEGF signalling controls the specification of endothelial cells to tip and stalk cells during angiogenesis sprouting. Alitalo and colleagues show that macrophage-derived VEGF-C activates VEGFR2 to contribute to the conversion of endothelial cells from a tip- to a stalk-cell fate when two sprouts fuse to ensure vessel growth and branching.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 2:2 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.

Placental angiogenesis is critical for normal development. Angiogenic factors and their receptors are key regulators of this process. Dysregulated placental vascular development is associated with pregnancy complications. Despite their importance, vascular growth factor expression has not been thoroughly correlated with placental morphologic development across gestation in cats. We postulate that changes in placental vessel morphology can be appreciated as consequences of dynamic expression of angiogenic signaling agents. Here, we characterized changes in placental morphology alongside expression analysis of angiogenic factor splice variants and receptors throughout pregnancy in domestic shorthair cats. We observed increased vascular and lamellar density in the lamellar zone during mid-pregnancy. Immunohistochemical analysis localized the vascular endothelial growth factor A (VEGF-A) receptor KDR to endothelial cells of the maternal and fetal microvasculatures. PlGF and its principal receptor Flt-1 were localized to the trophoblasts and fetal vasculature. VEGF-A was found in trophoblast cells and associated with endothelial cells. We detected expression of two Plgf splice variants and four Vegf-a variants. Quantitative real-time polymerase chain reaction analysis showed upregulation of mRNAs encoding pan Vegf-a and all Vegf-a splice forms at gestational days 30–35. Vegf-A showed a marked relative increase in expression during mid-pregnancy, consistent with the pro-angiogenic changes seen in the lamellar zone at days 30–35. Flt-1 was upregulated during late pregnancy. Plgf variants showed stable expression during the first two-thirds of pregnancy, followed by a marked increase toward term. These findings revealed specific spatiotemporal expression patterns of VEGF-A family members consistent with pivotal roles during normal placental development. Summary Sentence Morphological changes in the placenta during pregnancy correspond to distinctive expression patterns of vascular endothelial growth factor, placental growth factor, and their receptors, suggesting that placental morphologic dynamics are guided by differential expression of these angiogenic factors. Graphical Abstract

Vascular endothelial growth factors (VEGFs) have been shown to participate in atherosclerosis, arteriogenesis, cerebral edema, neuroprotection, neurogenesis, angiogenesis, postischemic brain and vessel repair, and the effects of transplanted stem cells in experimental stroke. Most of these actions involve VEGF-A and the VEGFR-2 receptor, but VEGF-B, placental growth factor, and VEGFR-1 have been implicated in some cases as well. VEGF signaling pathways represent important potential targets for the acute and chronic treatment of stroke.

We have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo, and this stimulation of nerve growth appears to be different from stimulation of angiogenesis by these same ligands, at least in part due to differences in VEGF receptor activation. VEGF signaling may be modulated by a number of factors including receptor number or the formation of receptor hetero- vs. homodimers. In endothelial cells, VEGF receptor heterodimer (VEGR1/R2) activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in neuronal cell types remains unclear. The purpose of this study was to identify the presence and distribution of VEGF Receptor-Ligand interactions in neuronal cells as compared to endothelial cells. PC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/μL) or VEGF-B (50 ng/μL) or \"vehicle\" (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) a third group of cells was also processed for Duolink Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF-receptor homo- and heterodimers in neuronal and endothelial cells. TIRF and fluorescence confocal microscopy revealed the presence of VEGFR1 co-localized with VEGFR2 in endothelial and PC12 neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in PC12 neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers as compared to endothelial cells whereas endothelial cells showed higher VEGFR2-R2 homodimers compared to neuronal cells as demonstrated by Duolink PLA. Levels of VEGFR1-R1 homodimers were very low in neuronal and endothelial cells. Differences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal versus endothelial cell types. This may in turn influence VEGF activity and regulation of neuronal cell homeostasis.

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Aflibercept is a recombinant fusion protein that acts as a soluble decoy receptor for vascular endothelial growth factor (VEGF), a key regulator of angiogenesis. It binds to all isoforms of VEGF-A as well as VEGF-B and placental growth factor, and, thus, prevents them from binding to and activating their cognate receptors. In the USA and EU, intravenously administered aflibercept in combination with an infusion of leucovorin, fluorouracil and irinotecan (FOLFIRI) is approved for the treatment of patients with metastatic colorectal cancer that is resistant to or has progressed after treatment with an oxaliplatin-containing regimen. The efficacy of aflibercept in this indication was assessed in a multinational, pivotal phase 3 trial (VELOUR), in which the approved regimen of aflibercept 4 mg/kg every 2 weeks plus FOLFIRI significantly prolonged median overall survival by 1.44 months compared with FOLFIRI alone (primary endpoint). The addition of aflibercept also significantly prolonged progression-free survival and significantly increased the objective response rate compared with FOLFIRI alone. Addition of aflibercept to FOLFIRI was associated with anti-VEGF-related adverse events and an increased incidence of FOLFIRI-related adverse events, but the tolerability of the combination was generally acceptable in this pre-treated population. The most common grade 3 or 4 adverse events with aflibercept plus FOLFIRI included neutropenia, diarrhoea and hypertension. In conclusion, aflibercept plus FOLFIRI is a useful treatment option for patients with metastatic colorectal cancer previously treated with an oxaliplatin-containing regimen, with or without bevacizumab.

Several studies support an important role of angiogenesis in breast cancer growth and metastasis. The main objectives of the study were to investigate the immunohistochemical expression of vascular endothelial growth factor (VEGF) family ligands (VEGF-A and VEGF-C) and receptors (VEGFR1, VEGFR2 and VEGFR3) in breast cancer and their associations with clinicopathological parameters, cancer subtypes/subgroups and patient outcome. Formalin-fixed paraffin-embedded tumor tissue samples were collected from early-stage breast cancer patients treated with anthracycline-based chemotherapy within a randomized trial. Immunohistochemistry was performed on serial 2.5 μm thick tissue sections from tissue microarray blocks. High VEGF-A, VEGF-C, VEGFR1, VEGFR2 and VEGFR3 protein expression was observed in 11.8% (N = 87), 80.8% (N = 585), 28.1% (N = 202), 64.6% (N = 359) and 71.8% (N = 517) of the cases, respectively. Significant associations were observed among all proteins (all p-values <0.05), with the exception of the one between VEGF-C and VEGFR1 (chi-square test, p = 0.15). Tumors with high VEGF-A protein expression, as compared to tumors with low expression were more frequently ER/PgR-negative (33.3% vs. 20.8%, chi-square test, p = 0.009) and HER2-positive (44.8% vs. 20.6%, p<0.001). In addition, tumors with high VEGFR1 expression, were more frequently HER2-positive (32.8% vs. 19.6%, p<0.001), while tumors with high VEGFR3 expression were more frequently ER/PgR-negative (24.9% vs. 17.0%, p = 0.024) and HER2-positive (26.9% vs. 14.8%, p = 0.001). High VEGF-A and VEGF-C protein expression was associated with increased DFS in the entire cohort (HR = 0.57, 95% CI 0.36-0.92, Wald's p = 0.020 and HR = 0.71, 95% CI 0.52-0.96, p = 0.025, respectively), as well as in specific subtypes/subgroups, such as HER2-positive (VEGF-A, HR = 0.32, 95% CI 0.14-0.74, p = 0.008) and triple-negative (VEGF-C, HR = 0.44, 95% CI 0.21-0.91, p = 0.027) patients. High vs. low VEGFR1 expression was an unfavorable factor for DFS in triple-negative patients (HR = 2.74, 95% CI 1.26-5.98, p = 0.011), whereas the opposite was observed among the ER/PgR-positive patients (HR = 0.69, 95% CI 0.48-0.98, p = 0.041). Regarding OS, high VEGF-C protein expression was associated with increased OS in the entire cohort (HR = 0.64, 95% CI 0.46-0.89, Wald's p = 0.008), as well as in in specific subtypes/subgroups, such as ER/PgR-negative (HR = 0.37, 95% CI 0.20-0.71, p = 0.003) and triple-negative (HR = 0.42, 95% CI 0.19-0.90, p = 0.026) patients. In conclusion, high expression of angiogenesis-related proteins is associated with adverse clinicopathological parameters in early-stage breast cancer patients and may be surrogate markers of biologically distinct subgroups of ER/PgR-negative or triple-negative tumors with superior outcome. Further validation of our findings in independent cohorts is needed.

Peripheral myopathy consists of a hallmark of heart failure (HF). Exercise enhanced skeletal muscle angiogenesis, and thus, it can be further beneficial towards the HF-induced myopathy. However, there is limited evidence regarding the exercise type that elicits optimum angiogenic responses of skeletal muscle in HF patients. This study aimed to (a) compare the effects of a high-intensity-interval-training (HIIT) or combined HIIT with strength training (COM) exercise protocol on the expression of angiogenesis-related factors in skeletal muscle of HF patients, and (b) examine the potential associations between the expression of those genes and capillarization in the trained muscles. Thirteen male patients with chronic HF (age: 51 ± 13 y; BMI: 27 ± 4 kg/m2) were randomly assigned to a 3-month exercise program that consisted of either HIIT (N = 6) or COM training (N = 7). Vastus lateralis muscle biopsies were performed pre- and post-training. RT-PCR was used to quantify the fold changes in mRNA expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), hypoxia-inducible factor 1 alpha (HIF-1α), angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2), angiopoietin receptor (Tie2), and matrix metallopeptidase 9 (MMP-9), and immunohistochemistry to assess capillarization in skeletal muscle post-training. There was an overall increase in the expression levels of VEGF, VEGFR-2, HIF-1α, Ang2, and MMP9 post-training, while these changes were not different among groups. Changes in capillary-to-fibre ratio were found to be strongly associated with Tie2 and HIF-1α expression. This was the first study demonstrating that both HIIT and combined HIIT with strength training enhanced similarly the expression profile of angiogenic factors in skeletal muscle of HF patients, possibly driving the angiogenic program in the trained muscles, although those gene expression increases were found to be only partially related with muscle capillarization.