Objective-Tissue factor (TF) initiates coagulation and indirectly triggers thrombin-dependent protease activated receptor (PAR) signaling. The TF-VIIa complex also directly cleaves PAR2 and promotes angiogenesis in vitro in TF cytoplasmic domain-deleted (TF ␦CT ) mice. Here we address the effect of PAR1 and PAR2 deficiency on angiogenesis in vivo. Methods and Results-In hypoxia-driven angiogenesis of oxygen induced retinopathy (OIR), wild-type, PAR1Ϫ/Ϫ , PAR2 Ϫ/Ϫ , and TF ␦CT mice showed a comparable regression of the superficial vascular plexus during the initial exposure of mice to hyperoxia. However, TF ␦CT mice revascularized areas of central vaso-obliteration significantly faster than wild-type animals. Pharmacological inhibition of the TF-VIIa complex, but not of Xa, and blockade of tyrosine kinase receptor pathways with Gleevec reversed accelerated angiogenesis of TF ␦CT mice to revascularization rates observed in wild-type mice. Genetic deletion of PAR2, but not of PAR1, abolished enhanced revascularization of TF ␦CT mice. PAR1 knock-out animals were indistinguishable from wild-type mice in the model of retinal neoangiogenesis and angiogenesis-dependent subcutaneous tumor growth was unaltered in PAR1-and PAR2-deficient animals. Key Words: tissue factor Ⅲ protease activated receptors Ⅲ angiogenesis Ⅲ coagulation Ⅲ thrombin T issue factor (TF), a transmembrane glycoprotein expressed by vascular and myeloid cells, is the primary cellular initiator of blood coagulation. TF exerts its biological activities by forming a catalytic enzyme complex with coagulation factor VIIa. The TF-VIIa complex then triggers coagulation by binding and activating factor X, leading to thrombin-dependent fibrin deposition and platelet activation. Thrombin can exhibit pleiotrophic effects which are typically mediated through G protein-coupled protease activated receptor (PAR) 1 signaling. 1 The TF-VIIa complex also signals directly by cleaving PAR2, but not PAR1. 2,3 However, the relative contributions of direct TF signaling, downstream coagulation activation, and indirect protease signaling to the underlying pathogenesis of cancer progression and angiogenesis, 3 are incompletely defined in vivo. Conclusion-LossTF deficiency results in vascular failure during development 4 and the proangiogenic phenotype of TF cytoplasmic domain deleted (TF ␦CT ) mice provided direct evidence that TF is involved in angiogenesis. 5 Whether direct TF-VIIa signaling is the only relevant pathway to drive angiogenesis in vivo has not been established. Indeed, thrombin-dependent PAR1 signaling stimulates angiogenesis in certain angiogenesis models in vivo, 6 -8 and the partial embryonic lethality of PAR1 knock-out mice attributable to vascular failure resembles the lethal phenotype of TF deficient animals. 9,10 However, the role of PAR1 in postnatal angiogenesis has not been directly addressed in PAR1 knock-out animals.Thrombin and/or TF-signaling pathways are also involved in tumor angiogenesis, 5,11 but production of proangiogenic factors by tumor cell...

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Skin wound closure occurs when keratinocytes migrate from the edge of the wound and re-epithelialize the epidermis. Their migration takes place primarily before any vascularization is established, that is, under hypoxia, but relatively little is known regarding the factors that stimulate this migration. Hypoxia and an acidic environment are well-established stimuli for cancer cell migration. The carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. On this basis, we explored the possible role of CAs in tissue regeneration using mouse skin wound models. We show that the expression of mRNAs encoding CA isoforms IV and IX are increased (~25 × and 4 ×, respectively) during the wound hypoxic period (days 2–5) and that cells expressing CAs form a band-like structure beneath the migrating epidermis. RNA-Seq analysis suggested that the CA IV-specific signal in the wound is mainly derived from neutrophils. Due to the high level of induction of CA IV in the wound, we treated skin wounds locally with recombinant human CA IV enzyme. Recombinant CA IV significantly accelerated wound re-epithelialization. Thus, CA IV could contribute to wound healing by providing an acidic environment in which the migrating epidermis and neutrophils can survive and may offer novel opportunities to accelerate wound healing under compromised conditions.

PurposeThe role of R-Ras in retinal angiogenesis and vascular permeability was evaluated in an oxygen-induced retinopathy (OIR) model using R-Ras knockout (KO) mice and in human diabetic neovascular membranes.MethodsMice deficient for R-Ras and their wild-type (WT) littermates were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. At P17 retinal vascularization was examined from whole mounts, and retinal vascular permeability was studied using Miles assay. Real-time RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of R-Ras in retina during development or in the OIR model. The degree of pericyte coverage and vascular endothelial (VE)-cadherin expression on WT and R-Ras KO retinal blood vessels was quantified using confocal microscopy. The correlation of R-Ras with vascular endothelial growth factor receptor 2 (VEGFR2) and human serum albumin on human proliferative diabetic retinopathy membranes was assessed using immunohistochemistry.ResultsIn retina, R-Ras expression was mostly restricted to the vasculature. Retinal vessels in the R-Ras KO mice were significantly more permeable than WT controls in the OIR model. A significant reduction in the direct physical contact between pericytes and blood vessel endothelium as well as reduced VE-cadherin immunostaining was found in R-Ras–deficient mice. In human proliferative diabetic retinopathy neovascular membranes, R-Ras expression negatively correlated with increased vascular leakage and expression of VEGFR2, a marker of blood vessel immaturity.ConclusionsOur results suggest that R-Ras has a role in controlling retinal vessel maturation and stabilization in ischemic retinopathy and provides a potential target for pharmacologic manipulation to treat diabetic retinopathy.

The results reveal new potential therapeutic targets to address hypoxia-induced pathological angiogenesis taking place in number of retinal diseases. The extensive proteomic profiling combined with pathway analysis also identifies novel molecular networks that could contribute to the pathogenesis of retinal diseases.

Retinal pigment epithelium (RPE) performs important functions for the maintenance of photoreceptors and vision. Malfunctions within the RPE are implicated in several retinal diseases for which transplantations of stem cell‐derived RPE are promising treatment options. Their success, however, is largely dependent on the functionality of the transplanted cells. This requires correct cellular physiology, which is highly influenced by the various ion channels of RPE, including voltage‐gated Ca2+ (CaV) channels. This study investigated the localization and functionality of CaV channels in human embryonic stem cell (hESC)‐derived RPE. Whole‐cell patch‐clamp recordings from these cells revealed slowly inactivating L‐type currents comparable to freshly isolated mouse RPE. Some hESC‐RPE cells also carried fast transient T‐type resembling currents. These findings were confirmed by immunostainings from both hESC‐ and mouse RPE that showed the presence of the L‐type Ca2+ channels CaV1.2 and CaV1.3 as well as the T‐type Ca2+ channels CaV3.1 and CaV3.2. The localization of the major subtype, CaV1.3, changed during hESC‐RPE maturation co‐localizing with pericentrin to the base of the primary cilium before reaching more homogeneous membrane localization comparable to mouse RPE. Based on functional assessment, the L‐type Ca2+ channels participated in the regulation of vascular endothelial growth factor secretion as well as in the phagocytosis of photoreceptor outer segments in hESC‐RPE. Overall, this study demonstrates that a functional machinery of voltage‐gated Ca2+ channels is present in mature hESC‐RPE, which is promising for the success of transplantation therapies. stem cells translational medicine 2019;8:179&15

AimsTo evaluate outcome of anti-vascular endothelial growth factor (VEGF) therapy for the treatment of neovascular age-related macular degeneration (nAMD) in the real-life setting and to compare incidence of ocular serious adverse events (SAE) after injections administered by nurses and physicians.MethodsRetrospective, single-centre study. Medical records of patients receiving anti-VEGF treatment for nAMD between 2008 and 2013 with three-loading-dose regimen were evaluated. Outcome measures were baseline visual acuity (VA), change in VA, number of intravitreal injections, incidence of ocular SAE and patients’ baseline characteristics affecting VA change. In addition, the number of injections per 1000 citizens living in the serving area and per individuals over 65 years old were estimated.Results1349 eyes in 1117 patients received a total of 11 562 intravitreal anti-VEGF injections. Twenty-one per cent of patients received treatment for both eyes. The mean baseline Snellen VA was 0.32. The mean change of VA from baseline was +2, +2 and ±0 Early Treatment Diabetic Retinopathy Study letters and the mean numbers of injections were 5.7, 4.7 and 4.9 at years 1, 2 and 3, respectively. There was a negative correlation between baseline VA and change of VA. Incidence of endophthalmitis was 0.086%. No difference in the incidence of ocular SAE was identified between injections given by nurses or by physicians. The number of intravitreal injections per all citizens was 9 per 1000 inhabitants and 45 per 1000 inhabitants over 65 years.ConclusionThe VA was maintained at the baseline level (±0 letters) with the mean of 15.3 anti-VEGF injections in real-world clinical practice during 3-year follow-up.

Oxygen-induced retinopathy (OIR) is a pure hypoxia-driven angiogenesis model and the most widely used model for ischemic retinopathies, such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). OIR model has been used to test new potential anti-angiogenic factors for human diseases. We have recently performed the most comprehensive characterization of OIR by a relatively novel mass spectrometry (MS) technique, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) proteomics and used genetically modified mice strains to identify novel molecular drug targets in angiogenic retinal diseases. We have confirmed the relevance of the identified molecular targets to human diseases by determining their expression pattern in neovascular membranes obtained from PDR and RVO patients. Based on our results, crystallins were the most prominent proteins induced by early hypoxic environment during the OIR, while actomyosin complex and Filamin A-R-Ras axis, that regulates vascular permeability of the angiogenic blood vessels, stood out at the peak of angiogenesis. Our results have revealed potential new therapeutic targets to address hypoxia-induced pathological angiogenesis and the associated vascular permeability in number of retinal diseases.