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Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3H2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.

International audience; Five vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds to VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.

The presence of α-synuclein aggregates in the retina of Parkinson’s disease patients has been associated with vision impairment. In this study we sought to determine the effects of α-synuclein overexpression on the survival and function of dopaminergic amacrine cells (DACs) in the retina. Adult mice were intravitreally injected with an adeno-associated viral (AAV) vector to overexpress human wild-type α-synuclein in the inner retina. Before and after systemic injections of levodopa (L-DOPA), retinal responses and visual acuity-driven behavior were measured by electroretinography (ERG) and a water maze task, respectively. Amacrine cells and ganglion cells were counted at different time points after the injection. α-synuclein overexpression led to an early loss of DACs associated with a decrease of light-adapted ERG responses and visual acuity that could be rescued by systemic injections of L-DOPA. The data show that α-synuclein overexpression affects dopamine neurons in the retina. The approach provides a novel accessible method to model the underlying mechanisms implicated in the pathogenesis of synucleinopathies and for testing novel treatments.

Placental growth factor (PlGF) is a proangiogenic member of the vascular endothelial growth factor (VEGF) family playing a central role in pathological angiogenesis. PlGF-DE is a PlGF variant unable to bind vascular endothelial growth factor receptor 1 (VEGFR-1) but still able to generate heterodimer with VEGF-A. We have generated PlGF-DE knockin mice that are vital and fertile and show unaltered expression of Plgf, Vegf-a, Vegfr-1, and Vegfr-2 compared with wild-type mice. Interestingly, these mutants showed additional and remarkable angiogenesis impairment in tumor growth, hindlimb ischemia, and choroidal neovascularization compared with both PlGF knockout and wild-type mice. These findings provided insights on VEGF-A/PlGF heterodimer function, which was able to rescue neovascularization and vascular leakage in PlGF-DE knockin mice. Collectively, these data show that PlGF-DE knockin mouse could be considered the full functional knockout of PlGF, suggesting a reassessment of the phenotypes of knockout mice for the genes whose products are able to generate heterodimeric proteins. Apicella et al. suggest a reconsideration of the phenotypes of knockout mice for molecular factors able to generate heterodimeric proteins. Knockin of Plgf, with a non-functional variant able to generate heterodimers with VEGF-A, determines additional pathological angiogenesis impairment compared with Plgf knockout, definitively proving the proangiogenic function of VEGF-A/PlGF heterodimers.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.

Eye drop formulations allowing topical treatment of retinal pathologies have long been sought as alternatives to intravitreal administration. This study aimed to assess whether a novel nanostructured microemulsions system (NaMESys) could be usefully employed to deliver sorafenib to the retina following topical instillation. NaMESys carrying 0.3% sorafenib (NaMESys-SOR) proved to be cytocompatible in vitro on rabbit corneal cells, and well-tolerated following b.i.d. ocular administration to rabbits during a 3-month study. In rats subject to retinal ischemia-reperfusion, NaMESys-SOR significantly inhibited retinal expression of tumor necrosis factor-alpha (TNFα, 20.7%) and inducible nitric oxide synthase (iNos, 87.3%) mRNAs in comparison to controls. Similarly, in streptozotocin-induced diabetic rats, NaMESys-SOR inhibited retinal expression of nuclear factor kappa B (NFκB), TNFα, insulin like growth factor 1 (IGF1), IGF1 receptor (IGF1R), vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2) mRNAs by three-fold on average compared to controls. Furthermore, a reduction in TNFα, VEGFR1 and VEGFR2 protein expression was observed by western blot. Moreover, in mice subject to laser-induced choroidal neovascularization, NaMESys-SOR significantly inhibited neovascular lesions by 54%. In conclusion, NaMESys-SOR was shown to be a well-tolerated ophthalmic formulation able to deliver effective amounts of sorafenib to the retina, reducing proinflammatory and pro-angiogenic mediators in reliable models of proliferative retinopathies. These findings warrant further investigations on the full therapeutic potential of NaMESys-SOR eye drops, aiming to address unmet needs in the pharmacotherapy of retinal neovascular diseases.

Helper T cells actively communicate with adjacent cells by secreting soluble mediators, yet crosstalk between helper T cells and endothelial cells remains poorly understood. Here we found that placental growth factor (PlGF), a homolog of the vascular endothelial growth factor that enhances an angiogenic switch in disease, was selectively secreted by the TH17 subset of helper T cells and promoted angiogenesis. Interestingly, the 'angio-lymphokine' PlGF, in turn, specifically induced the differentiation of pathogenic TH17 cells by activating the transcription factor STAT3 via binding to its receptors and replaced the activity of interleukin-6 in the production of interleukin-17, whereas it suppressed the generation of regulatory T cells. Moreover, T cell-derived PlGF was required for the progression of autoimmune diseases associated with TH17 differentiation, including experimental autoimmune encephalomyelitis and collagen-induced arthritis, in mice. Collectively, our findings provide insights into the PlGF-dictated links among angiogenesis, TH17 cell development and autoimmunity.

Diabetic retinopathy (DR) is a secondary complication of diabetes. DR can cause irreversible blindness, and its pathogenesis is considered multifactorial. DR can progress from non-proliferative DR to proliferative DR, characterized by retinal neovascularization. The main cause of vision loss in diabetic patients is diabetic macular edema, caused by vessel leakage and blood retinal barrier breakdown. Currently, aflibercept is an anti-VEGF approved for diabetic macular edema. Aflibercept can bind several members of vascular permeability factors, namely VEGF-A, B, and PlGF. We analyzed the aflibercept-PlGF complex at molecular level, through an in silico approach. In order to explore the role of PlGF in DR, we treated primary human retinal endothelial cells (HRECs) and mouse retinal epithelial cells (RPEs) with aflibercept and an anti-PlGF antibody. We explored the hypothesis that aflibercept has anti-inflammatory action through blocking of PlGF signaling and the ERK axis in an in vitro and in vivo model of DR. Both aflibercept and the anti-PlGF antibody exerted protective effects on retinal cells, by inhibition of the ERK pathway. Moreover, aflibercept significantly decreased (p < 0.05) the expression of TNF-? in an in vitro and in vivo model of DR. Therefore, our data suggest that inhibition of PlGF signaling, or a selective blocking, may be useful in the management of early phases of DR when the inflammatory process is largely involved.

Combinatorially generated molecular repertoires have been largely used to identify novel bioactive compounds. Ever more sophisticated technological solutions have been proposed to simplify and speed up such process, expanding the chemical diversity space and increasing the prospect to select new molecular entities with specific and potent activities against targets of therapeutic relevance. In this context, random mixtures of oligomeric peptides were originally used and since 25 years they represent a continuous source of bioactive molecules with potencies ranging from the sub-nM to microM concentration. Synthetic peptide libraries are still employed as starting “synthetic broths” of structurally and chemically diversified molecular fragments from which lead compounds can be extracted and further modified. Thousands of studies have been reported describing the application of combinatorial mixtures of synthetic peptides with different complexity and engrafted on diverse structural scaffolds for the identification of new compounds which have been further developed and also tested in in vivo models of relevant diseases. We briefly review some of the most used methodologies for library preparation and screening and the most recent case studies appeared in the literature where compounds have reached at least in vivo testing in animal or similar models. Recent technological advancements in biotechnology, engineering and computer science have suggested new options to facilitate the discovery of new bioactive peptides. In this instance, we anticipate here a new approach for the design of simple but focused tripeptide libraries against druggable cavities of therapeutic targets and its complementation with existing approaches.

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Hypoxia plays a crucial role in the angiogenic switch, modulating a large set of genes mainly through the activation of hypoxia-inducible factor (HIF) transcriptional complex. Endothelial cells play a central role in new vessels formation and express placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, mainly involved in pathological angiogenesis. Despite several observations suggest a hypoxia-mediated positive modulation of PlGF, the molecular mechanism governing this regulation has not been fully elucidated. We decided to investigate if epigenetic modifications are involved in hypoxia-induced PlGF expression. We report that PlGF expression was induced in cultured human and mouse endothelial cells exposed to hypoxia (1% O 2), although DNA methylation at the Plgf CpG-island remains unchanged. Remarkably, robust hyperacetylation of histones H3 and H4 was observed in the second intron of Plgf, where hypoxia responsive elements (HREs), never described before, are located. HIF-1?, but not HIF-2?, binds to identified HREs. Noteworthy, only HIF-1? silencing fully inhibited PlGF upregulation. These results formally demonstrate a direct involvement of HIF-1? in the upregulation of PlGF expression in hypoxia through chromatin remodeling of HREs sites. Therefore, PlGF may be considered one of the putative targets of anti-HIF therapeutic applications.

The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low μm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders1-4. But the molecular underpinnings of the avascular phenotype have until now remained obscure5-10 and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap11 by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/− mice12,13 and Pax6+/− patients with aniridia14 are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/− mice. Manatees, the only known creatures uniformly to have vascularized corneas15, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in a variety of functions in vertebrates, especially angiogenesis. Here we identify and characterize a PDGF/VEGF-like factor (named PVF-1) from the nematode C. elegans. We show that PVF-1 has biochemical properties similar to vertebrate PDGF/VEGF growth factors. More important, PVF-1 binds to the human receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) and is able to induce angiogenesis in two model systems derived from vertebrates. Our results highlight the widespread evolutionary conservation of this important class of growth factors and raise the possibility that C. elegans can provide a simple experimental system in which to investigate how these factors function.—Tarsitano, M., De Falco, S., Colonna, V., McGhee, J. D., Persico, M. G. The C. elegans pvf-1 gene encodes a PDGF/VEGF-like factor able to bind mammalian VEGF receptors and to induce angiogenesis.

Background Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery. Methods and Results We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction. Conclusion PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers.

Neovascular age-related macular degeneration is a leading cause of irreversible vision loss in the Western world. Cytokine-targeted therapies (such as anti-vascular endothelial growth factor) are effective in treating pathologic ocular angiogenesis, but have not led to a durable effect and often require indefinite treatment. Here, we show that Nutlin-3, a small molecule antagonist of the E3 ubiquitin protein ligase MDM2, inhibited angiogenesis in several model systems. We found that a functional p53 pathway was essential for Nutlin-3-mediated retinal antiangiogenesis and disruption of the p53 transcriptional network abolished the antiangiogenic activity of Nutlin-3. Nutlin-3 did not inhibit established, mature blood vessels in the adult mouse retina, suggesting that only proliferating retinal vessels are sensitive to Nutlin-3. Furthermore, Nutlin-3 inhibited angiogenesis in nonretinal models such as the hind limb ischemia model. Our work demonstrates that Nutlin-3 functions through an antiproliferative pathway with conceivable advantages over existing cytokine-targeted antiangiogenesis therapies.

Summary Exploitation of embryonic stem cells (ESC) for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat), a class I histone deacetylase inhibitor (HDAC), modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.

The proangiogenic members of the vascular endothelial growth factor (VEGF) family and related receptors play a central role in the modulation of pathological angiogenesis. In order to identify plant compounds able to interfere in the VEGFs/VEGFR-1 (Flt-1) recognition by VEGF family members, the extracts of the aerial parts of Campsiandra guayanensis and Feretia apodanthera were screened by a competitive ELISA-based assay. By using this bioassay-oriented approach five proanthocyanindins, including the new natural compounds (2S)-4',5,7-trihydroxyflavan-(4?->8)-afzelechin (1) and (2S)-4',5,7-trihydroxyflavan-(4?->8)-epiafzelechin (2) and the known geranin B (3), proanthocyanidin A2 (4), and proanthocyanidin A1 (5), were isolated. The study of the antiangiogenic activities of compounds 1-5 using ELISA and SPR assays showed compound 1 as being the most active. The antiangiogenic activity of 1 was also confirmed in vivo by the chicken chorioallantoic membrane assay. Our results indicated 1 as a new antiangiogenic compound inhibiting the interaction between VEGF-A or PlGF and their receptor VEGRF-1.

Angiogenesis is a crucial step in many pathological conditions like cancer, inflammation and metastasis formation; on these basis the search for antiangiogenic agents has widened. In order to identify new compounds able to interfere in the Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1, also known as Flt-1) recognition by VEGFs family members, we screened Calycolpus moritzianus (O. Berg) Burret leaves extracts by a competitive ELISA-based assay. MeOH and CHCl3 extracts and several their fractions demonstrated to be able to prevent VEGF or PlGF interaction with Flt-1, with an inhibition about 50% at concentration of 100 μg/mL. Phytochemical and pharmacological investigation of the active fractions led to the isolation of flavonoids, and terpenes.

Angiogenesis is one of the crucial events for cancer development and growth. Two members of the vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if coexpressed in the same cell, are both required for pathologic angiogenesis. We have generated a PlGF1 variant, named PlGF1-DE in which the residues Asp72 and Glu73 were substituted with Ala, which is unable to bind and activate VEGF receptor-1 but is still able to heterodimerize with VEGF. Here, we show that overexpression in tumor cells by adenoviral delivery or stable transfection of PlGF1-DE variant significantly reduces the production of VEGF homodimer via heterodimerization, determining a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel lumen and stabilization, and monocyte-macrophage infiltration. Conversely, the overexpression of PlGF1wt, also reducing the VEGF homodimer production comparably with PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, does not inhibit tumor growth and vessel density compared with controls but induces increase of vessel lumen, vessel stabilization, and monocyte-macrophage infiltration. The property of PlGF and VEGF-A to generate heterodimer represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a “dominant negative” of VEGF and is a new candidate for antiangiogenic gene therapy in cancer treatment. Cancer Res; 70(5); 1804–13

Objective To delineate the role of neuropilin-1 (NP-1), a vascular endothelial growth factor receptor (VEGFR), in rheumatoid inflammation and to determine whether blockade of NP-1 could suppress synoviocyte survival and angiogenesis. Methods VEGF(111-165) peptide, which encompasses the NP-1 binding domain of VEGF(165), was generated by cleaving VEGF(165) with plasmin. The effect of this peptide on the interaction between VEGF(165) and its receptor was determined by (125)I-VEGFR binding assay. Assays to determine synoviocyte apoptosis, adhesion, and migration were performed in the presence of VEGF(165) and/or the peptide. VEGF(165)-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of endothelial cells (ECs). Mice were immunized with type II collagen to induce experimental arthritis. Results VEGF(111-165) peptide specifically inhibited the binding of (125)I-VEGF(165) to NP-1 on rheumatoid synoviocytes and ECs. The peptide eliminated the VEGF(165)-mediated increase in synoviocyte survival and activation of p-ERK and Bcl-2. The peptide also completely inhibited a VEGF(165)-induced increase in synoviocyte adhesion and migration. In addition, the anti-NP-1 peptide blocked VEGF(165)-stimulated proliferation, capillary tube formation, and wounding migration of ECs in vitro. VEGF(165)-induced neovascularization in a Matrigel plug in mice was also blocked by treatment with the peptide. Finally, subcutaneous injection of anti-NP-1 peptide suppressed arthritis severity and autoantibody formation in mice with experimental arthritis and inhibited synoviocyte hyperplasia and angiogenesis in arthritic joints. Conclusion Anti-NP-1 peptide suppressed VEGF(165)-induced increases in synoviocyte survival and angiogenesis, and thereby blocked experimental arthritis. Our findings suggest that anti-NP-1 peptide could be useful in alleviating chronic arthritis.

Aims Flt-1 is an fms-like tyrosine kinase receptor which binds to vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Ligand activation and blocking of flt-1 influence several vascular smooth muscle cell (SMC) functions, including apoptotic susceptibility. However, downstream signal transduction pathways by which flt-1 regulates SMC apoptosis have still to be investigated. Methods and results Flt-1 expression and apoptosis in Wistar rat aortic intimal cells 15 days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, western blotting, and PCR. Anti-flt1 blocking antibody effects were compared with those of anti-PlGF and anti-VEGF antibodies. Rat aortic intimal cells 15 days after injury exhibited increased flt-1 protein and mRNA and lower smooth muscle markers compared with normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC(50) = 16.5 ng/mL) and anti-PlGF (EC(50) = 20.5 ng/mL) antibodies added to intimal cultures reduced serum-deprived apoptosis but not serum-and PDGF-BB-induced proliferation; the anti-VEGF antibody was ineffective. Sorted flt-1(+) cells were more clonogenic than flt-1 and whole intimal SMC populations. Increased nuclear factor-kappaB (NF-kappa B) and inhibitor of apoptosis protein-1 (IAP-1) and reduced bax levels associated with the anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-kappa B activity inhibitor and IAP-1 interfering RNA (RNAi). Blocking of NF-kappa B activity reduced IAP-1 expression and prevented IAP-1 RNAi effects. Increased flt-1 immunoreaction was also documented in human atheromatous lesions. Conclusion Our results show that anti-flt-1 blocking reduces apoptosis through NF-kappa B and the downstream IAP-1 pathway. The close link between flt-1, PlGF, and apoptotic susceptibility of intimal SMCs suggests new potential strategies aimed at influencing post-injury arterial remodelling.

The activation of vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is crucial in many physiological and pathological conditions, like angiogenesis, cancer, inflammation, hematopoiesis, bone marrow precursors/stem cells recruitment in tumor angiogenesis, and metastasis formation. Many recent reports indicate that molecules able to antagonize Flt-1 activity have gained a strong interest in the view of therapeutic approaches. In order to identify new compounds able to interfere in the Flt-1 recognition by VEGFs family members, we have developed a highly sensitive competitive ELISA-based screening to study plant extracts and derivatives. Several fractions of the N-butanol extract of Pteleopsis suberosa leaves and of the chloroform extract of Parinari campestris leaves demonstrated by a bioassay-guided fractionation an evident inhibition of VEGF-A or placental growth factor (PlGF) interaction with Flt-1, with an inhibition over 50 % in particular for the VEGF-A/Flt-1 interaction at a concentration of 100 microg/mL. This activity seems be due to the presence of a combination of compounds acting synergistically.

Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC50 ∼ 10 μm). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role.

Neo-angiogenesis is a complex phenomenon modulated by the concerted action of several molecular factors. We have generated a congenic line of knockout mice carrying null mutations of both placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS), two genes that play a pivotal role in the regulation of pathological angiogenesis. In the present study, we describe the phenotype of this new experimental animal model after surgically induced hind-limb ischemia. Plgf–/–, eNos–/–, Plgf–/– eNos–/–, and wild-type C57BL/6J mice were studied. Plgf–/– eNos–/– mice showed the most severe phenotype: self-amputation, and death occurred in up to 47% of the animals studied; in ischemic legs, capillary density was severely reduced; macrophage infiltration and oxidative stress increased as compared to the other groups of animals. These changes were associated with an up-regulation of both inducible NOS (iNOS) expression and vascular endothelial growth factor (VEGF) protein levels in ischemic limbs, and to an increased extent of protein nitration. Our results demonstrate that the deletion of these two genes, Plgf, which acts in synergism with VEGF, and eNos, a downstream mediator of VEGF, determines a significant change in the vascular response to an ischemic stimulus and that oxidative stress within the ischemic tissue represents a crucial factor to maintain tissue homeostasis.—Gigante, B., Morlino, G., Gentile, M. T., Persico, M. G., De Falco, S. Plgf–/–eNos–/– mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia.

We report for the first time the chemical synthesis of refolded CFC domain of mouse Cripto (mCFC) and of two variants bearing mutations on residues W107 and H104 involved in Alk4 binding. The domains undergo spontaneous and quantitative refolding in about 4 h, yet with very different kinetics. Bisulfide linkages have been assessed by enzyme digestion and mass spectrometry analysis of resulting fragments, and the first experimental studies on structural organization have been conducted by circular dichroism spectroscopy under different pH conditions. Upon refolding, the domains considerably change their conformations, although they do not assume canonical structures, and become highly resistant to enzyme degradation. A comparative study of receptor binding shows that the CFC domain can bind Alk4 and confirms the importance of W107 and H104 for receptor recognition. © 2006 Wiley-Liss, Inc.

Placenta growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in pathological conditions. PlGF exerts its biological function through the binding and activation of the seven immunoglobulin-like domain receptor Flt-1, also known as VEGFR-1. Here, we report the first detailed mutagenesis studies that provide a basis for understanding molecular recognition between PlGF-1 and Flt-1, highlighting some of the residues that are critical for receptor recognition. Mutagenesis analysis, performed on the basis of a structural model of interaction between PlGF and the minimal binding domain of Flt-1, has led to the identification of several PlGF-1 residues involved in Flt-1 recognition. The two negatively charged residues, Asp-72 and Glu-73, located in the beta3-beta4 loop, are critical for Flt-1 binding. Other mutations, which bring about a significant decrease in PlGF binding activity, are Gln-27, located in the N-terminal alpha-helix, and Pro-98 and Tyr-100 on the beta6 strand. The mutation of one of the two glycosylated residues of PlGF, Asn-84, generates a PlGF variant with reduced binding activity. This indicates that, unlike in VEGF, glycosylation plays an important role in Flt-1 binding. The double mutation of residues Asp-72 and Glu-73 generates a PlGF variant unable to bind and activate the receptor molecules on the cell surface. This variant failed to induce in vitro capillary-like tube formation of primary endothelial cells or neo-angiogenesis in an in vivo chorioallantoic membrane assay.

Angiogenesis is a tightly regulated process, both during development and adult life. Animal models with mutations in the genes coding for placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, or the tyrosine kinase domain of the PlGF receptor (Flt-1) have revealed differences between normal physiological angiogenesis and pathological angiogenesis associated with conditions such as tumor growth, arthritis and atherosclerosis. In the present paper, we investigated the potential role of PlGF in regulating physiological angiogenesis by analyzing vascular changes in heart and skeletal muscles of wild-type and Plgf−/− mice following prolonged and sustained physical training. Sedentary Plgf−/− mice showed a reduced capillary density in both heart and skeletal muscles as compared to wild-type mice (P < 0.05). However, after a 6-week training period, heart/body weight ratio, citrate synthase activity, vessel density and capillary/myocyte ratio were significantly increased in both wild-type and Plgf−/− mice (all P < 0.05). At the same time intercapillary distance was significantly reduced. Finally, acute exercise was not associated with any change in PlGF protein level in the skeletal muscle. Our results demonstrate that PlGF is not necessary for exercise-training-induced angiogenesis. We thus suggest that the role of PlGF is confined to the selective regulation of angiogenesis only under pathological conditions.

Amidating mouse pituitary cells (AtT-20) have been engineered to secrete human calcitonin (hCT) in the fully active amidated form, without the need of additional enzymatic or chemical modifications. The 141-residue human calcitonin precursor has first been cloned in the eucaryotic expression vector pRc/RSV, and the resulting plasmid pRc/RSV/hCT introduced in AtT-20 cells. After transfection, 122 independent clones resistant to G-418 were selected and screened for calcitonin production using a competitive ELISA specifically designed to detect the amidated form of calcitonin. One of these clones was amplified and showed expression of 17 ng/ml of hCT, with a 70% increase in productivity after cAMP treatment. Calcitonin was partially purified from culture medium by two sequential steps of reverse-phase chromatography and characterized in terms of immunoreactivity and molecular weight by TOF-MALDI mass spectroscopy, which confirmed the intended chemical nature and the presence of the C-terminal amidated residue.

Angiogenesis is one of the crucial events for cancer development and growth. Two members of vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if co-expressed in the same cell, are both required for pathological angiogenesis. We generated a PlGF1 variant, named PlGF1-DE in which the residues Asp72 and Glu73 were substituted with Ala. PlGF1-DE is unable to bind and activate VEGFR-1 but can heterodimerize with VEGF (J. Biol. Chem., 279: 43929-43939, 2004). Here we demonstrate that overexpression of PlGF1-DE variant in tumor cells by adenoviral delivery or stable transfection significantly reduced the production of VEGF homodimer via heterodimerization, leading to a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel size, vessel stabilization by smooth muscle cells and monocyte-macrophage infiltration. PlGF1-DE/VEGF heterodimer, differently from wild type heterodimer able to induce VEGFR-1 dimerization as well as VEGFR-1/R-2 heterodimerization and activation, failed to induce receptors phosphorylation demonstrating that co-expression of PlGF1-DE variant in VEGF-producing cells effectively sequestered active VEGF, biasing toward the generation of a non-functional heterodimer. Conversely, overexpression of PlGF1wt, while reducing VEGF homodimer production comparably to PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, did not inhibit tumor growth and vessel density compared to controls, but induced an increase of vessel size, vessel stabilization and monocyte-macrophage infiltration. The ability of PlGF and VEGF-A to heterodimerize represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a ‘dominant negative’ of VEGF molecule and is a new candidate for anti-angiogenic gene therapy in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-370.

In order to direct antisense oligonucleotides to specific tissues or cell types in vivo, we are exploring the possibility to utilize lipoproteins as transport vehicles. A 16-mer oligonucleotide (ODN) was derivatized at the 5' prime through a 32P phosphate spacer with cholesterol, yielding a 32P-labeled amphiphatic cholesteryl-oligonucleotide (cholODN). Incubation of cholODN with low-density lipoprotein (LDL) for 2 hr at 37 degrees C resulted in the formation of a cholODN-LDL complex that migrates as a single peak on agarose gel electrophoresis. The cholODN was found to bind quantitatively to both high-density lipoproteins (HDL) and LDL, but not to albumin. Stable oligonucleotide-LDL particles with up to 50 molecules of cholODN per LDL particle could be obtained. In contrast, the control ODN did not show affinity for plasma lipoproteins. Upon injection into rats, cholODN became rapidly associated with plasma lipoproteins while control ODNs were recovered in the lipoprotein deficient serum fraction. The plasma half-life of cholODN (9-11 min) is considerably prolonged as compared with the control ODN (t1/2 less than 1 min). The cholODN-LDL was at least 5 min stable against degradation by rat plasma nucleases. It is concluded that derivatization of antisense oligonucleotides with cholesterol profoundly modifies their in vivo fate and opens possibilities for efficient and specific receptor-dependent targeting, mediated by lipoproteins coupled with specific recognition markers to various hepatic cell types.