Placental growth factor regulates the generation of TH17 cells to link angiogenesis with autoimmunity.

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.

Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1.

In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling. The purified and bioconjugated molecules have been extensively characterized and tested on Cripto-1-positive cancer cells through in vitro binding assays. These recombinant Fab fragments recognize the target antigen in its native form on intact cells suggesting that they can be further developed as reagents for detecting Cripto-1 in theranostic settings.

Synergistic interactions of PlGF and VEGF contribute to blood-retinal barrier breakdown through canonical NFκB activation.

To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) heterodimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF heterodimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naïve animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF heterodimers, VEGF, PlGF, NFκB, p-IκBα, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-IκBα, and colocalization of NFκB p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, PlGF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pIκBα. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NFκB activation.

Prolyl 3-Hydroxylase 2 Is a Molecular Player of Angiogenesis.

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.

Assessment of a New Nanostructured Microemulsion System for Ocular Delivery of Sorafenib to Posterior Segment of the Eye.

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.

Oral Delivery of a Tetrameric Tripeptide Inhibitor of VEGFR1 Suppresses Pathological Choroid Neovascularization.

Age-related macular degeneration (AMD) is the primary cause of blindness in advanced countries. Repeated intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents has represented an important advancement for the therapy of wet AMD with significative results in terms of blindness prevention and partial vision restore. Nonetheless, some patients are not responsive or do not attain significant visual improvement, intravitreal injection may cause serious complications and important side effects have been reported for the prolonged block of VEGF-A. In order to evaluate new anti-angiogenic strategies, we focused our attention on VEGF receptor 1 (VEGFR1) developing a specific VEGFR-1 antagonist, a tetrameric tripeptide named inhibitor of VEGFR 1 (iVR1). We have evaluated its anti-angiogenic activity in the preclinical model of AMD, the laser-induced choroid neovascularization (CNV). iVR1 is able to potently inhibit CNV when delivered by intravitreal injection. Surprisingly, it is able to significantly reduce CNV also when delivered by gavage. Our data show that the specific block of VEGFR1 in vivo represents a valid alternative to the block of VEGF-A and that the inhibition of the pathological neovascularization at ocular level is also possible by systemic delivery of compounds not targeting VEGF-A.

Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications.

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.

Hedgehog signalling pathway orchestrates angiogenesis in triple-negative breast cancers.

BACKGROUND: Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated. METHODS: Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice. RESULTS: Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation. CONCLUSIONS: This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

Placental growth factor-1 and -2 induce hyperplasia and invasiveness of primary rheumatoid synoviocytes.

Inflammation-mediated oncogenesis has been implicated in a variety of cancer types. Rheumatoid synovial tissues can be viewed as a tumor-like mass, consisting of hyperplastic fibroblast-like synoviocytes (FLSs). FLSs of rheumatoid arthritis (RA) patients have promigratory and invasive characteristics, which may be caused by chronic exposure to genotoxic stimuli, including hypoxia and growth factors. We tested whether a transformed phenotype of RA-FLSs is associated with placental growth factor (PlGF), a representative angiogenic growth factor induced by hypoxia. In this study, we identified PlGF-1 and PlGF-2 as the major PlGF isoforms in RA-FLSs. Global gene expression profiling revealed that cell proliferation, apoptosis, angiogenesis, and cell migration were mainly represented by differentially expressed genes in RA-FLSs transfected with small interfering RNA for PlGF. Indeed, PlGF-deficient RA-FLSs showed a decrease in cell proliferation, migration, and invasion, but an increase in apoptotic death in vitro. PlGF gene overexpression resulted in the opposite effects. Moreover, exogeneous PlGF-1 and PlGF-2 increased survival, migration, and invasiveness of RA-FLSs by binding their receptors, Flt-1 and neuropilin-1, and upregulating the expression of antiapoptotic molecules, pErk and Bcl2. Knockdown of PlGF transcripts reduced RA-FLS proliferation in a xenotransplantation model. Collectively, in addition to their role for neovascularization, PlGF-1 and -2 promote proliferation, survival, migration, and invasion of RA-FLSs in an autocrine and paracrine manner. These results demonstrated how primary cells of mesenchymal origin acquired an aggressive and transformed phenotype. PlGF and its receptors thus offer new targets for anti-FLS therapy.

Powerful tumor cell growth-inhibiting activity of a synthetic derivative of atractyligenin: involvement of PI3K/Akt pathway and thioredoxin system.

BACKGROUND: The semi-synthetic ent-kaurane 15-ketoatractyligenin methyl ester (SC2017) has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. Our study was aimed at investigating SC2017 tumor growth-inhibiting activity and the underlying mechanisms in Jurkat cells (T-cell leukemia) and xenograft tumor models. METHODS: Cell viability was evaluated by MTT assay. Cell cycle progression, reactive oxygen species (ROS) elevation and apoptotic hallmarks were monitored by flow cytometry. Inhibition of thioredoxin reductase (TrxR) by biochemical assays. Levels and/or activation status of signaling proteins were assessed by western blotting. Xenograft tumors were generated with HCT 116 colon carcinoma cells. RESULTS: SC2017 displayed cell growth-inhibiting activity against Jurkat cells (half maximal inhibitory concentration values (IC50)<2µM), but low cell-killing potential in human peripheral blood mononuclear cells (PBMC). The primary response of Jurkat cells to SC2017 was an arrest in G2 phase followed by caspase-dependent apoptosis. Inhibition of PI3K/Akt pathway and TrxR activity by SC2017 was demonstrated by biochemical and pharmacological approaches. At least, SC2017 was found to inhibit xenograft tumor growth. CONCLUSIONS: Our results demonstrate that SC2017 inhibits tumor cell growth in in vitro and in vivo models, but displays moderate toxicity against PBMC. We also demonstrate that SC2017 promotes caspase-dependent apoptosis in Jurkat cells by affecting Akt activation status and TrxR functionality. GENERAL SIGNIFICANCE: Our observations suggest the semi-synthetic ent-kaurane SC2017 as a promising chemotherapeutic compound. SC2017 has, indeed, shown to possess tumor growth inhibiting activity and be able to counteract PI3K/Akt and Trx system survival signaling.

PlGF and VEGF-A/PlGF Heterodimer are Crucial for Recruitment and Activation of Immune Cells During Choroid Neovascularization.

Purpose: Recruitment and activation of inflammatory cells, such as retinal microglia/macrophages, in the subretinal space contribute significantly to the pathogenesis of age-related macular degeneration (AMD). This study aims to explore the functional role of vascular endothelial growth factor (VEGF-A), placental growth factor (PlGF) and VEGF-A/PlGF heterodimer in immune homeostasis and activation during pathological laser-induced choroidal neovascularization (CNV). Methods: To investigate these roles, we utilized the PlGF-DE knockin (KI) mouse model, which is the full functional knockout (KO) of PlGF. In this model, mice express a variant of PlGF, named PlGF-DE, that is unable to bind and activate VEGFR-1 but can still form heterodimer with VEGF-A. Results: Our findings demonstrate that, although there is no difference in healthy conditions, PlGF-DE-KI mice exhibit decreased microglia reactivity and reduced recruitment of both microglia and monocyte-macrophages, compared to wild-type mice during laser-induced CNV. This impairment is associated with a reduction in VEGF receptor 1 (VEGFR-1) phosphorylation in the retinae of PlGF-DE-KI mice compared to C57Bl6/J mice. Corroborating these data, intravitreal delivery of PlGF or VEGF-A/PlGF heterodimer in PlGF-DE-KI mice rescued the immune cell response at the early phase of CNV compared to VEGF-A delivery. Conclusions: In summary, our study suggests that targeting PlGF and the VEGF-A/PlGF heterodimer, thereby preventing VEGFR-1 activation, could represent a potential therapeutic approach for the management of inflammatory processes in diseases such as AMD.

Cancer-derived exosomal Alu RNA promotes colorectal cancer progression.

Inflammation plays a crucial role in cancer progression, but the relevance of the inflammasome remains unclear. Alu RNA was the first endogenous nucleic acid shown to activate the NLRP3 (nucleotide-binding domain leucine-rich repeat containing 3) inflammasome. Here, we showed that Alu RNA can induce epithelial-to-mesenchymal transition (EMT) through NLRP3 inflammasome activation and IL-1ß release in colorectal cancer (CRC) cells. Alu RNA is stored, transported and transferred to CRC cells by exosomes. Exosomal Alu RNA promotes tumorigenesis by inducing invasion, metastasis and EMT via NLRP3 inflammasome activation. Consistent with these data, we found that significantly increased Alu RNA expression correlates with the induction of NLRP3 priming in human CRC patients. Furthermore, the level of Alu RNA in circulating exosomes correlates with CRC progression in a preclinical model. These findings reveal the direct involvement of Alu RNA in cancer pathogenesis, and its presence in CRC cell-derived exosomes could be used as a noninvasive diagnostic biomarker.

Bioassay-guided isolation of proanthocyanidins with antiangiogenic activities.

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-(4ß→8)-afzelechin (1) and (2S)-4',5,7-trihydroxyflavan-(4ß→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.

The biflavonoid amentoflavone inhibits neovascularization preventing the activity of proangiogenic vascular endothelial growth factors.

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.

Corneal avascularity is due to soluble VEGF receptor-1.

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 disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure 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 trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia 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 corneas, 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.

Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth.

Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.

A small synthetic cripto blocking Peptide improves neural induction, dopaminergic differentiation, and functional integration of mouse embryonic stem cells in a rat model of Parkinson's disease.

Cripto is a glycosylphosphatidylinositol-anchored coreceptor that binds Nodal and the activin type I (ALK)-4 receptor, and is involved in cardiac differentiation of mouse embryonic stem cells (mESCs). Interestingly, genetic ablation of cripto results in increased neuralization and midbrain dopaminergic (DA) differentiation of mESCs, as well as improved DA cell replacement therapy (CRT) in a model of Parkinson's disease (PD). In this study, we developed a Cripto specific blocking tool that would mimic the deletion of cripto, but could be easily applied to embryonic stem cell (ESC) lines without the need of genetic manipulation. We thus screened a combinatorial peptide library and identified a tetrameric tripeptide, Cripto blocking peptide (BP), which prevents Cripto/ALK-4 receptor interaction and interferes with Cripto signaling. Cripto BP treatment favored neuroectoderm formation and promoted midbrain DA neuron differentiation of mESCs in vitro and in vivo. Remarkably, Cripto BP-treated ESCs, when transplanted into the striatum of PD rats, enhanced functional recovery and reduced tumor formation, mimicking the effect of genetic ablation of cripto. We therefore suggest that specific blockers such as Cripto BP may be used to improve the differentiation of ESC-derived DA neurons in vitro and their engraftment in vivo, bringing us closer towards an application of ESCs in CRT.

Propionyl-L-carnitine improves postischemic blood flow recovery and arteriogenetic revascularization and reduces endothelial NADPH-oxidase 4-mediated superoxide production.

OBJECTIVE: The beneficial effect of the natural compound propionyl-l-carnitine (PLC) on intermittent claudication in patients with peripheral arterial disease is attributed to its anaplerotic function in ischemic tissues, but inadequate information is available concerning action on the vasculature. METHODS AND RESULTS: We investigated the effects of PLC in rabbit hind limb collateral vessels after femoral artery excision, mouse dorsal air pouch, chicken chorioallantoic membrane, and vascular cells by angiographic, Doppler flow, and histomorphometrical and biomolecular analyses. PLC injection accelerated hind limb blood flow recovery after 4 days (P<0.05) and increased angiographic quadriceps collateral vascularization after 7 days (P<0.001) Histomorphometry confirmed the increased vascular area (P<0.05), with unchanged intramuscular capillary density. PLC-induced dilatative adaptation, and growth was found associated with increased inducible nitric oxide synthase and reduced arterial vascular endothelial growth factor and intracellular adhesion molecule-1 expression. PLC also increased vascularization in air pouch and chorioallantoic membrane (P<0.05), particularly in large vessels. PLC increased endothelial and human umbilical vascular endothelial cell proliferation and rapidly reduced inducible nitric oxide synthase and NADPH-oxidase 4-mediated reactive oxygen species production in human umbilical vascular endothelial cells; NADPH-oxidase 4 also regulated NF-kappaB-independent intracellular adhesion molecule-1 expression. CONCLUSIONS: Our results provided strong evidence that PLC improves postischemic flow recovery and revascularization and reduces endothelial NADPH-oxidase-related superoxide production. We recommend that PLC should be included among therapeutic interventions that target endothelial function.

VEGFR1 signaling in retinal angiogenesis and microinflammation.

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 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.

Long Non-coding RNA T-UCstem1 Controls Progenitor Proliferation and Neurogenesis in the Postnatal Mouse Olfactory Bulb through Interaction with miR-9.

Neural stem cell populations generate a wide spectrum of neuronal and glial cell types in a highly ordered fashion. MicroRNAs are essential regulators of this process. T-UCstem1 is a long non-coding RNA containing an ultraconserved element, and in vitro analyses in pluripotent stem cells provided evidence that it regulates the balance between proliferation and differentiation. Here we investigate the in vivo function of T-UCstem1. We show that T-UCstem1 is expressed in the forebrain neurogenic lineage that generates interneurons for the postnatal olfactory bulb. Gain of function in neural stem cells increased progenitor proliferation at the expense of neuron production, whereas knockdown had the opposite effect. This regulatory function is mediated by its interaction with miR-9-3p and miR-9-5p. Based thereon, we propose a mechanistic model for the role of T-UCstem1 in the dynamic regulation of neural progenitor proliferation during neurogenesis.