VEGF Family Members Research Articles

Effcacy of a chimeric PlGF derived protein for the management of preeclampsia

Preeclampsia (PE) is one of the most common complications of pregnancy, affecting ~5-7% of pregnancies. Hallmarked by new onset hypertension and maternal endothelial dysfunction, PE is believed to be caused by insuffcient blood flow to the placenta. This causes the release of factors into the maternal circulation; notably the soluble VEGF receptor sFlt-1, which antagonizes the effects of VEGF in the endothelium. Placental growth factor (PlGF) is a VEGF family member produced by the placenta that only binds to the Flt-1 receptor and has little inherent angiogenic activity. We hypothesized that administration of a stabilized chimeric PlGF-derived protein (ELP-PlGF) would antagonize sFlt-1 in a rodent model of PE, and help resolve the maternal symptoms. We therefore utilized the reduced uterine perfusion pressure (RUPP) model of placental ischemia. RUPP animals exhibited a significant increase in blood presssure (99 vs 118 mmHg p<005), while RUPP animals treated with ELP-PlGF demonstrated significantly attenuated hypertension (105mmHg, p<0.05). Likewise, RUPP animals treated with ELP-PlGF showed decreased placental sFlt-1 (512 vs 308 pg/mg, p<0.05), and plasma sFlt-1 by western blot (13877 vs 6860 AU, p<0.05). Further, vascular preproendothelin expression from the aorta decreased 60% between RUPP and RUPP treated animals. Taken together, these results suggest that PlGF-based therapeutics could be a viable approach to the management of PE, and this formulation demonstrates significant effcacy in vivo in a relevant animal model. This work was partially supported by NIH grants P01HL51971, P20GM104357, and R01HL137791. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of lymphatic vessels in corneal fluid homeostasis and wound healing.

The cornea, essential for vision, is normally avascular, transparent, and immune-privileged. However, injuries or infections can break this privilege, allowing blood and lymphatic vessels to invade, potentially impairing vision and causing immune responses. This review explores the complex role of corneal lymphangiogenesis in health and diseases. Traditionally, the cornea was considered devoid of lymphatic vessels, a phenomenon known as "corneal (lymph)angiogenic privilege." Recent advances in molecular markers have enabled the discovery of lymphatic vessels in the cornea under certain conditions. Several molecules contribute to preserving both immune and lymphangiogenic privileges. Lymphangiogenesis, primarily driven by VEGF family members, can occur directly or indirectly through macrophage recruitment. Corneal injuries and diseases disrupt these privileges, reducing graft survival rates following transplantation. However, modulation of lymphangiogenesis offers potential interventions to promote graft survival and expedite corneal edema resolution.This review underscores the intricate interplay between lymphatic vessels, immune privilege, and corneal pathologies, highlighting innovative therapeutic possibilities. Future investigations should explore the modulation of lymphangiogenesis to enhance corneal health and transparency, as well as corneal graft survival, and this benefits patients with various corneal conditions.

Erythropoietin receptor is a risk factor for prognosis: A potential biomarker in lung adenocarcinoma

Lung cancer has the highest mortality rate of all cancers, and LUAD's survival rate is particularly poor. Erythropoietin receptor (EPOR) can be detected in lung adenocarcinoma (LUAD), however, the expression levels and prognostic value of EPOR in LUAD are still unclear. In our study, clinicopathological data of 92 LUAD patients between January 2008 and June 2016, multiple bioinformatics databases and immunohistochemistry were used to explore the EPOR expression, the mutant genes affecting EPOR expression, and the correlation of EPOR expression with oxidative stress - related genes, prognosis, immune microenvironment. All statistical analyses were performed in the R version 4.1.1. The study found that EPOR expression might be down-regulated at the mRNA levels and significantly up-regulated at the protein levels in LUAD, which indicates that the mRNA and protein levels of EPOR are inconsistent. The muTarget showed that the expression of EPOR was significantly different between the mutant group and the wild group of 15 genes, including DDX60L and C1orf168. Importantly, we found that EPOR was associated with VEGF and HIF family members, and had significant positive correlation with oxidative stress - related genes such as CCS, EPX and TXNRD2. This suggests that EPOR may be involved in the regulation of oxidative stress. The Kaplan-Meier Plotter and PrognoScan databases consistently concluded that EPOR was associated with prognosis in LUAD patients. Our clinicopathological data showed that high EPOR expression was associated with poorer overall survival (29.5 vs 46 months) and had a good predictive ability for 4-year and 5-year survival probability. EPOR is expected to be a potential new prognostic marker for LUAD.

VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

A VEGFB-Based Peptidomimetic Inhibits VEGFR2-Mediated PI3K/Akt/mTOR and PLCγ/ERK Signaling and Elicits Apoptotic, Antiangiogenic, and Antitumor Activities.

Vascular endothelial growth factor receptor 2 (VEGFR2) mediates VEGFA signaling mainly through the PI3K/AKT/mTOR and PLCγ/ERK1/2 pathways. Here we unveil a peptidomimetic (VGB3) based on the interaction between VEGFB and VEGFR1 that unexpectedly binds and neutralizes VEGFR2. Investigation of the cyclic and linear structures of VGB3 (named C-VGB3 and L-VGB3, respectively) using receptor binding and cell proliferation assays, molecular docking, and evaluation of antiangiogenic and antitumor activities in the 4T1 mouse mammary carcinoma tumor (MCT) model showed that loop formation is essential for peptide functionality. C-VGB3 inhibited proliferation and tubulogenesis of human umbilical vein endothelial cells (HUVECs), accounting for the abrogation of VEGFR2, p-VEGFR2 and, subsequently, PI3K/AKT/mTOR and PLCγ/ERK1/2 pathways. In 4T1 MCT cells, C-VGB3 inhibited cell proliferation, VEGFR2 expression and phosphorylation, the PI3K/AKT/mTOR pathway, FAK/Paxillin, and the epithelial-to-mesenchymal transition cascade. The apoptotic effects of C-VGB3 on HUVE and 4T1 MCT cells were inferred from annexin-PI and TUNEL staining and activation of P53, caspase-3, caspase-7, and PARP1, which mechanistically occurred through the intrinsic pathway mediated by Bcl2 family members, cytochrome c, Apaf-1 and caspase-9, and extrinsic pathway via death receptors and caspase-8. These data indicate that binding regions shared by VEGF family members may be important in developing novel pan-VEGFR inhibitors that are highly relevant in the pathogenesis of angiogenesis-related diseases.

Genetic variants of vascular endothelial growth factor-634 and vascular endothelial growth factor-936 in Circassians and Chechens subpopulations in Jordan

BackgroundVascular endothelial growth factor (VEGF) is a signaling protein that promotes the growth of new blood vessels in vasculogenesis and angiogenesis. The most important member of VEGF family is VEGF-A which bind to VEGFR-1 and VEGFR-2 and plays a major role in diseases that involve blood vessels such as Tumor Angiogenesis, Age-related Macular Degeneration (AMD) and Diabetic Retinopathy (DR). ObjectiveStudying the interindividual variability in VEGF by determining the allele frequency for certain genetic polymorphisms of VEGF-936 and VEGF-634 genes in two subpopulations in Jordan; Circassians and Chechens, as well as comparing the allele frequencies with other populations, including Jordanian Arabs. Methods319 unrelated healthy Circassian and Chechen individuals were genotyped for VEGF-936 and VEGF-634 by using PCR and RFLP. ResultsWe found that Circassians did not have any significant difference in allele frequencies of VEGF-634 compared to the Jordanian Arab population and all three populations had similar frequencies of VEGF-936. ConclusionThese findings provide genetic information that may serve as a basis for larger studies designed to assess variability associated with VEGF polymorphisms. They also provide important data for the implementation of personalized medicine in Circassians and Chechens populations living in Jordan.

VEGF and VEGFR family members are expressed by neoplastic cells of NF1-associated tumors and may play an oncogenic role in malignant peripheral nerve sheath tumor growth through an autocrine loop

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. The role of angiogenesis and VEGF pathway in the pathogenesis of neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) remains poorly understood.We assessed the expression of VEGF and VEGFR family members in cohorts of plexiform neurofibromas (pNF), MPNSTs and MPNST cell lines at transcript [pNF, n = 49; MPNST, n = 34] and protein levels [pNF, n = 21; MPNST, n = 9].VEGF and VEGFR members were variably expressed in cell lines. VEGFA (p = 3.10−5), VEGFR1 (p = 0.08), and VEGFR2 (p = 2.10−4) mRNAs were overexpressed in MPNSTs in comparison with pNFs. Both VEGFA and VEGFR1 proteins were expressed by spindle tumor cells of pNFs and MPNSTs. VEGFA was expressed more in MPNSTs than in pNFs (p = 9.10−6) and a trend for VEGFR1 overexpression was observed (p = 0.06). VEGFR2 was not found at the protein level. The microvascular density was significantly reduced in MPNSTs as compared to pNFs (p = 0.0025), with no differences regarding the expression of the activated phosphorylated forms of ERK (P-ERK [p = 0.63]) and AKT (P-AKT [p = 0.41]) in endothelial cells, suggesting that VEGF-dependant angiogenesis may not be critical for MPNST oncogenesis.Altogether, these results indicate that the VEGF-VEGFR pathway may play a role in the development of pNFs and MPNSTs, independently of angiogenesis. Whether or not it drives an oncogenic autocrine/paracrine loop in neoplastic cells, participating in an increased activation of signaling pathways downstream of tyrosine kinase receptors, including VEGFRs, is a tempting hypothesis. Nevertheless, the specific targeting of angiogenesis in MPNSTs may not be sufficient to slow down tumor growth.

The significance of PlGF in the progression of the ovarian hyperstimulation syndrome in patient undergoing an in vitro fertilization procedure

Background: Placenta growth factor is, VEGF family member, a Pleiotropic Factor affects a variety of cell types and regulates various biological responses. PlGF is similar in structure and function to VEGF, and it amplifies VEGF's angiogenic actions. Ovulation stimulation during cycles of in vitro fertilization (IVF) for the mangment of infertility results in ovarian hyperstimulation syndrome (OHSS), an iatrogenic side effect. Considering potential roles of PlGF and its receptor (sflt-1) in angiogenesis, the association of these factors with OHSS among study women during controlled ovarian stimulation have evaluated in the present study. Methods: comparative cross sectional study including of 60 women who go through controlled ovarian stimulation. On oocyte retrieval day, their serum and follicular fluid were taken. The concentrations of PlGF and sFlt-1 were assessed using ELISA. Results: Eighteen patients presented with ovarian hyperstimulation syndrome (OHSS) and 42 patients were no OHSS. There was significantly higher serum PIGF in women with OHSS patients as compared to women with no OHSS, 141.4 ± 11.8 versus 91.9 ± 5.4 respectively (p<0.001). Furthermore Follicular fluid PlGF there was significantly higher in women with OHSS patients as compared to women with no OHSS, 163.4 ± 7.2 versus 91.1 ± 5.5 respectively (p<0.001).additionally Follicular fluids PlGF/sFlt ratio significantly higher in women with OHSS patients as compared to women with no OHSS, 0.034 ± 0.01 versus 0.026 ± 0.01 respectively (p<0.001). On the contrary there was significantly lower serum and follicular fluid sFlt in ovarian hyper-stimulated patients Conclusions: These statistics show that serum and FF PlGF and PlGF to sFlt ratio higher in women with ovarian hyperstimulation syndrome (OHSS) patients as compared to women with no OHSS. So the PlGF may be played an important a role in angiogenesis dysregulation.

Gene expression profile of the murine ischemic retina and its response to Aflibercept (VEGF-Trap)

Ischemic retinal dystrophies are leading causes of acquired vision loss. Although the dysregulated expression of the hypoxia-responsive VEGF-A is a major driver of ischemic retinopathies, implication of additional VEGF-family members in their pathogenesis has led to the development of multivalent anti-angiogenic tools. Designed as a decoy receptor for all ligands of VEGFR1 and VEGFR2, Aflibercept is a potent anti-angiogenic agent. Notwithstanding, the molecular mechanisms mediating Aflibercept’s efficacy remain only partially understood. Here, we used the oxygen-induced retinopathy (OIR) mouse as a model system of pathological retinal vascularization to investigate the transcriptional response of the murine retina to hypoxia and of the OIR retina to Aflibercept. While OIR severely impaired transcriptional changes normally ensuing during retinal development, analysis of gene expression patterns hinted at alterations in leukocyte recruitment during the recovery phase of the OIR protocol. Moreover, the levels of Angiopoietin-2, a major player in the progression of diabetic retinopathy, were elevated in OIR tissues and consistently downregulated by Aflibercept. Notably, GO term, KEGG pathway enrichment, and expression dynamics analyses revealed that, beyond regulating angiogenic processes, Aflibercept also modulated inflammation and supported synaptic transmission. Altogether, our findings delineate novel mechanisms potentially underlying Aflibercept’s efficacy against ischemic retinopathies.

Placental growth factor exerts modulatory effects on in vitro activated T cells

Background . Recent studies demonstrated immunosuppressive properties of vascular endothelial growth factor (VEGF-A) and identified VEGF-A as a key mediator of tumor-induced immunosuppression. Placental growth factor (PlGF) is another member of VEGF family in which dramatic elevation is associated with effective immune adaptation in successful pregnancy, whereas low concentrations are related to pregnancy complications resulting from the activation of immune system. Previously, we have shown that activated T cells express VEGF receptor type 1 (VEGFR-1), and PlGF inhibits T cell proliferation in VEGFR-1–dependent manner. The aim of the present study was to further characterize PlGF effects on T cell responses in vitro . Materials and methods. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with anti-CD3 monoclonal antibodies (a-CD3) in the absence or presence of PlGF and assessed for IL-10 production, programmed cell death and the expression of inhibitory receptors (PD-1, CTLA-4, Tim-3) in CD4+ and CD8+ T cell subsets. Results. The addition of PlGF to PBMC cultures activated with a-CD3 resulted in increased percentages of IL- 10-producing CD4+ and CD8+ T cells. Besides, PlGF promoted CD8+ T cells apoptosis while did not affect programmed cell death within CD4+ T cells. Notable, T cell activation with a-CD3 in the presence of PlGF was accompanied by the enhancement of PD-1-expressing cells in CD8+ T cell subset and Tim-3-expressing cells in both CD4+ and CD8+ T cells, and by the increased expression of PD-1 and Tim-3 on T cells. Conclusion. Our in vitro findings indicate that PlGF can inhibit T cell responses due to the increasing interleukin-10 (IL-10) production, promoting CD8+ T cell apoptosis and enhancing the expression of PD-1 and Tim-3 inhibitory receptors. Given the elevated levels of PlGF in successful pregnancy and its decrease in gestation complications, the obtained data also suggest that PlGF-mediated suppression may be implicated into the governing immune evasion in pregnancy.

The binding of kaempferol-3-O-rutinoside to vascular endothelial growth factor potentiates anti-inflammatory efficiencies in lipopolysaccharide-treated mouse macrophage RAW264.7 cells

BackgroundVascular Endothelial Growth Factors (VEGFs) are a group of growth factor in regulating development and maintenance of blood capillary. The VEGF family members include VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. VEGF receptor activation leads to multiple complex signaling pathways, particularly in inducing angiogenesis. Besides, VEGF is produced by macrophages and T cells, which is playing roles in inflammation. In macrophages, VEGF receptor-3 (VEGFR-3) and its ligand VEGF-C are known to attenuate the release of pro-inflammatory cytokines. MethodsImmunoprecipitation and molecular docking assays showed the binding interaction of kaempferol-3-O-rutinoside and VEGF-C. Western blotting and qRT-PCR methods were applied to explore the potentiating effect of kaempferol-3-O-rutinoside in VEGF-C-mediated expressions of proteins and genes in endothelial cells and LPS-induced macrophages. Enzyme-linked immunosorbent assay (ELISA) was employed to reveal the release of proinflammatory cytokines in LPS-induced macrophages. Immunofluorescence assay was performed to determine the effect of kaempferol-3-O-rutinoside in regulating nuclear translocation of NF-κB p65 subunit in the VEGF-C-treated cultures. In addition, Transwell® motility assay was applied to detect the ability of cell migration after drug treatment in LPS-induced macrophages. ResultsWe identified kaempferol-3-O-rutinoside, a flavonoid commonly found in vegetable and fruit, was able to act on cultured macrophages in inhibiting inflammatory response, and the inhibition was mediated by its specific binding to VEGF-C. The kaempferol-3-O-rutinoside-bound VEGF-C showed high potency to trigger the receptor activation. In LPS-treated cultured macrophages, applied kaempferol-3-O-rutinoside potentiated inhibitory effects of exogenous applied VEGF-C on the secretions of pro-inflammatory cytokines, i.e. IL-6 and TNF-α, as well as expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). This inhibition was in parallel to transcription and translocation of NF-κB. Moreover, the binding of kaempferol-3-O-rutinoside with VEGF-C suppressed the LPS-induced migration of macrophage. ConclusionTaken together, our results suggested the pharmacological roles of kaempferol-3-O-rutinoside in VEGF-C-mediated anti-inflammation.

VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions.

PurposeMüller glia are important in retinal health and disease and are a major source of retinal VEGF-A. Of the different VEGF family members, the role of VEGF-A in retinal health and disease has been studied extensively. The potential contribution of other VEGF family members to retinal pathophysiology, however, remains poorly defined. This study aimed to understand the role of VEGF-B in Müller cell pathophysiology.MethodsThe expression of different VEGFs and their receptors in human MIO-M1 and mouse QMMuC-1 Müller cell lines and primary murine Müller cells was examined by RT-PCR, ELISA, and Western blot. The effect of recombinant VEGF-B or VEGF-B neutralization on Müller cell viability and survival under normal, hypoxic, and oxidative (4-hydroxynonenal [4-HNE]) conditions was evaluated by Alamar Blue, Yo-Pro uptake, and immunocytochemistry. The expression of glial fibrillary acidic protein, aquaporin-4, inward rectifying K+ channel subtype 4.1, glutamine synthetase, and transient receptor potential vanilloid 4 under different treatment conditions was examined by RT-PCR, immunocytochemistry, and Western blot. Transient receptor potential vanilloid 4 channel activity was assessed using a Fura-2–based calcium assay.ResultsVEGF-B was expressed in Müller cells at the highest levels compared with other members of the VEGF family. VEGF-B neutralization did not affect Müller cell viability or functionality under normal conditions, but enhanced hypoxia– or 4-HNE–induced Müller cell death and decreased inward rectifying K+ channel subtype 4.1 and aquaporin-4 expression. Recombinant VEGF-B restored Müller cell glutamine synthetase expression under hypoxic conditions and protected Müller cells from 4-HNE–induced damage by normalizing transient receptor potential vanilloid 4 channel expression and activity.ConclusionsAutocrine production of VEGF-B protects Müller cells under pathologic conditions.

Факторы семейства VEGF, IGF и TGF-β1 в ткани сальника при раке яичников

Факторы семейства VEGF, IGF и TGF-β1 в ткани сальника при раке яичников

Ranibizumab Alters Levels of Intraocular Soluble Cytokine Receptors in Patients with Diabetic Macular Edema

ABSTRACTPurpose: Ranibizumab, an anti-VEGF-A (vascular endothelial cell growth factor-A) monoclonal antibody fragment, is a well-established treatment for diabetic patients with macular edema. However, very little is known about the effect of ranibizumab on intraocular regulation of pro – and anti-inflammatory signaling pathways and their regulation of VEGF family members, which was the aim of this study.Materials and Methods: Diabetic patients (n = 10) aged ≥18 years with central diabetic macular edema, BCVA >24 and <78, and central macular thickness (CMT) greater than 250 μm were enrolled in this study. Following a full eye exam, imaging, and an aqueous tap, patients received ranibizumab (0.3 mg/0.05 mL) injections at day one and weeks four and eight. At week 12, a full eye exam, imaging, and a second aqueous tap was obtained prior to the last injection of ranibizumab. Pre – and post-treatment aqueous humor samples were then analyzed using Milliplex MAP magnetic bead assays.Results: As expected, ranibizumab lowered levels of VEGF-A, decreased CMT, and improved VA (visual acuity). In addition, it significantly lowered aqueous levels of IL-10, IFNγ, sIL-1R1, sIL-1R2, sRAGE, and VEGF-D. Changes in levels of VEGF-A and VEGF-C strongly correlated with changes in soluble receptors, sgp130 and sIL-6R, associated with IL-6 signaling pathways. In contrast, changes in VEGF-D correlated with sIL-1R1 and sIL-1R2, soluble receptors participating in IL-1 signaling. Changes in CMT and VA did not correlate with changes in levels of VEGF family members. However, post-treatment values of CMT correlated with post-treatment levels of VEGF-C. Post-treatment VA values correlated with a wide variety of potential biomarkers linked to inflammation.Conclusions: Ranibizumab treatment had strong effects on regulating levels of soluble receptors closely linked to IL-1 and IL-6 signaling pathways. Therefore, a complete understanding of the actions of ranibizumab will further the development of additional therapies to support treatment of diabetic macular edema.

Defining the Inflammatory Microenvironment in the Human Cochlea by Perilymph Analysis: Toward Liquid Biopsy of the Cochlea.

The molecular pathomechanisms in the majority of patients suffering from acute or progressive sensorineural hearing loss cannot be determined yet. The size and the complex architecture of the cochlea make biopsy and in-depth histological analyses impossible without severe damage of the organ. Thus, histopathology correlated to inner disease is only possible after death. The establishment of a technique for perilymph sampling during cochlear implantation may enable a liquid biopsy and characterization of the cochlear microenvironment. Inflammatory processes may not only participate in disease onset and progression in the inner ear, but may also control performance of the implant. However, little is known about cytokines and chemokines in the human inner ear as predictive markers for cochlear implant performance. First attempts to use multiplex protein arrays for inflammatory markers were successful for the identification of cytokines, chemokines, and endothelial markers present in the human perilymph. Moreover, unsupervised cluster and principal component analyses were used to group patients by lead cytokines and to correlate certain proteins to clinical data. Endothelial and epithelial factors were detected at higher concentrations than typical pro-inflammatory cytokines such as TNF-a or IL-6. Significant differences in VEGF family members have been observed comparing patients with deafness to patients with residual hearing with significantly reduced VEGF-D levels in patients with deafness. In addition, there is a trend toward higher IGFBP-1 levels in these patients. Hence, endothelial and epithelial factors in combination with cytokines may present robust biomarker candidates and will be investigated in future studies in more detail. Thus, multiplex protein arrays are feasible in very small perilymph samples allowing a qualitative and quantitative analysis of inflammatory markers. More results are required to advance this method for elucidating the development and course of specific inner ear diseases or for perioperative characterization of cochlear implant patients.

Novel multi-targeted inhibitors suppress ocular neovascularization by regulating unique gene sets

Neovascular diseases, such as many cancers and ocular disorders, are life threatening and devastating. Although anti-vascular endothelial growth factor A (VEGF-A) therapy is available, many patients are not responsive and drug resistance can develop. To try to overcome these problems, combination therapy targeting VEGF-A and platelet-derived growth factor B (PDGF-B) was tested. However, one obvious drawback was that the other VEGF and PDGF family members were not inhibited and therefore could compensate. Indeed, this was, at least to some extent, demonstrated by the disappointing outcomes. To this end, we designed novel multi-targeted inhibitors that can block most of the VEGF and PDGF family members simultaneously by making a fusion protein containing the ligand-binding domains of vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor beta (PDGFRβ), which can therefore act as a decoy blocker for most of the VEGF and PDGF family members. Indeed, in cultured cells, the novel inhibitors suppressed the migration and proliferation of both vascular endothelial cells and smooth muscle cells, and abolished VEGFR2 and PDGFRβ activation. Importantly, in a choroidal neovascularization model in vivo, the novel inhibitor inhibited ocular neovascularization more efficiently than the mono-inhibitors against VEGFR or PDGFR alone respectively. Mechanistically, a genome-wide microarray analysis unveiled that the novel inhibitor regulated unique sets of genes that were not regulated by the mono-inhibitors, further demonstrating the functional uniqueness and superiority of the novel inhibitor. Together, we show that the multi-targeted inhibitors that can block VEGFR1, VEGFR2 and PDGFRβ simultaneously suppress pathological angiogenesis more efficiently than monotherapy, and may therefore have promising therapeutic value for the treatment of neovascular diseases.

Short PlGF-derived peptides bind VEGFR-1 and VEGFR-2 in vitro and on the surface of endothelial cells.

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR-1), which occurs predominantly through receptor domains 2 and 3. The PlGF β-hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR-1, but replacement of G94 with residues bearing H-bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF-2) and G94W (peptide PlGF-10) mutants, while the peptide PlGF-8, bearing the R96G mutation, is totally inactive. The PlGF-1 and PlGF-2 peptides also bind the VEGFR-2 receptors, though with a reduced affinity, and are able to interfere with the VEGF-induced receptor signaling on endothelial cells. The peptides also bind VEGFR-2 on the surface of cells, while PlGF-8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.

Plasma levels of VEGF-A, VEGF B, and VEGFR-1 and applicability of these parameters as tumor markers in diagnosis of breast cancer.

The VEGF family members are important factors in promoting angiogenesis and lymphangiogenesis in malignant processes. The aim of this study was to investigate plasma concentrations of VEGF-A, VEGF-B and their soluble VEGFR-1 receptor and their diagnostic utility and potency as compared to CA 15-3 in breast cancer patients and in relation to the control group. The study included 120 breast cancer patients and 60 control patients. Plasma levels of tested parameters were determined with ELISA and CA 15-3 levels were determined with CMIA. Concentrations of all tested parameters in breast cancer patients showed statistically significant difference when compared to the control groups (benign breast tumor patients and/or healthy women). VEGF-B showed the highest values of sensitivity (Sn) and predictive value of a negative test result (NPV) in total BC group (90% and 66.7%, respectively) and, more importantly, in stages I-II of BC (SE: 86.8%; 92.7%, NPV: 82.8%; 88.9%, respectively). Among all parameters tested, VEGF-A showed the highest specificity (Sf) (76.7%) and predictive value of a positive test result (PPV) (84.8%), yet they were lower than for CA 15-3. VEGF-A was also the best parameter that had statistically significant Area Under Curve (AUC) in stages I (0.678) and II (0.768). In the whole group of BC patients all parameters tested showed statistically significant AUC, but the maximum range was obtained for the combination of VEGF-A and CA 15-3 (0.817). The combined analysis of the studied parameters and CA 15-3 resulted in an increase in sensitivity and AUC values, which provides hope for developing a new panel of biomarkers that may be used in BC diagnosis in the future.

TRP Channels in Angiogenesis and Other Endothelial Functions.

Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial organization of endothelial cells. This mechanism is regulated by a balance between stimulatory and inhibitory factors. Proangiogenic factors include a variety of VEGF family members, while thrombospondin and endostatin, among others, have been reported as suppressors of angiogenesis. Transient receptor potential (TRP) channels belong to a superfamily of cation-permeable channels that play a relevant role in a number of cellular functions mostly derived from their influence in intracellular Ca2+ homeostasis. Endothelial cells express a variety of TRP channels, including members of the TRPC, TRPV, TRPP, TRPA, and TRPM families, which play a relevant role in a number of functions, including endothelium-induced vasodilation, vascular permeability as well as sensing hemodynamic and chemical changes. Furthermore, TRP channels have been reported to play an important role in angiogenesis. This review summarizes the current knowledge and limitations concerning the involvement of particular TRP channels in growth factor-induced angiogenesis.

Human Plasma Levels of VEGF-A, VEGF-C, VEGF-D, their Soluble Receptor - VEGFR-2 and Applicability of these Parameters as Tumor Markers in the Diagnostics of Breast Cancer

VEGF family members are important factors in promoting angio- and lymphangiogenesis. The aim of this study was to investigate concentrations, diagnostic utility and power of VEGF-A, VEGF-C, VEGF-D and VEGFR-2 in comparison to CA15–3 in breast cancer (BC) patients. The study included 120 BC patients and 60 control patients (28 with benign breast tumors and 32 healthy women). Plasma levels of tested parameters were determined by ELISA, CA15–3 by CMIA. Concentrations of all parameters showed statistical significance when compared BC patients to controls. VEGF-D showed the highest SE (82.50%) in total BC group. Highest SP and PPV in total BC group showed VEGF-A(76.67%;84.78%,respectively), but lower than CA15–3. Highest NPV showed VEGF-C(52.33%), but it was lower than CA15–3. VEGF-C was also the best parameter which had statistically significant AUC in total cancer group (0.7672), but also stages I(0.7684) and II(0.7772). In the total group of BC almost all tested parameters showed statistically significant AUC, but a maximum range was obtained for the combination of VEGF-C + CA15–3(0.8476). The combined analysis of tested parameters and CA15–3 resulted in increase in SE and AUC values, which provides hope for developing a new panel of biomarkers that may be used in the diagnosis of BC in the future.