Blocking Ocular Sympathetic Activity Inhibits Choroidal Neovascularization

Purpose: To investigate how modulating ocular sympathetic activity affects progression of choroidal neovascularization (CNV), a hallmark feature of wet age-related macular degeneration (AMD).Methods: In the first of two studies, Brown Norway rats underwent laser-induced CNV and were assigned to one of the following groups: daily eye drops of artificial tears (n = 10; control group); daily eye drops of the β-adrenoreceptor agonist isoproterenol (n = 10); daily eye drops of the β-adrenoreceptor antagonist propranolol (n = 10); sympathetic internal carotid nerve (ICN) transection 6 weeks prior to laser-induced CNV (n = 10). In the second study, rats underwent laser-induced CNV followed by ICN transection at different time points: immediately after the laser injury (n = 6), 7 days after the laser injury (n = 6), and sham surgery 7 days after the laser injury (n = 6; control group). All animals were euthanized 14 days after laser application. CNV development was quantified with fluorescein angiography and optical coherence tomography (in vivo), as well as lesion volume analysis using 3D confocal reconstruction (postmortem). Angiogenic growth factor protein levels in the choroid were measured with ELISA.Results: In the first study, blocking ocular sympathetic activity through pharmacological or surgical manipulation led to a 75% or 70% reduction in CNV lesion volume versus the control group, respectively (P < 0.001). Stimulating ocular sympathetic activity with isoproterenol also led to a reduction in lesion volume, but only by 27% versus controls (P < 0.05). VEGF protein levels in the choroid were elevated in the three treatment groups (P < 0.01). In the second study, fluorescein angiography and CNV lesion volume analysis indicated that surgically removing the ocular sympathetic supply inhibited progression of laser-induced CNV, regardless of whether ICN transection was performed on the same day or 7 days after the laser injury.Conclusion: Surgical and pharmacological block of ocular sympathetic activity can inhibit progression of CNV in a rat model. Therefore, electrical block of ICN activity could be a potential bioelectronic medicine strategy for treating wet AMD.

Investigating the Roles of Platelet PAR4 in Hemostasis, Thrombosis and Viral Infection Using a Newly Generated PAR4 Floxed Mouse

Introduction: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes and lung epithelial cells, and activated by multiple ligands including thrombin and cathepsin G. Importantly, PAR4 is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and protection from thrombosis which are attributed to loss of platelet PAR4, but this has not been specifically demonstrated in mice. Additionally, global PAR4 deficiency increases mortality after influenza A virus infection, but the cell type/s responsible for the enhanced mortality have not been determined. Here, we describe the generation of PAR4 floxed (PAR4 fl/fl) mice that can be used to delete PAR4 in a cell type-specific manner, and examine the effect of megakaryocyte/platelet-specific deletion of PAR4 on hemostasis, thrombosis and viral infection using PAR4 fl/fl;PF4Cre + mice. Methods: PAR4global knockout (PAR4 -/-), MK/platelet-specific knockout (PAR4 fl/fl;PF4Cre +) and appropriate littermate control mice were used for experiments. Platelet function was determined by light transmission aggregometry and flow cytometry. Hemostasis was assessed in the saphenous vein laser injury model. Platelet plug formation was visualized by intravital microscopy following saphenous vein laser ablation (~50 μm diameter injury), followed by 2 subsequent ablations to reinjure the same site unless on-going bleeding was occurring. Mice were treated with ibrutinib (12.5 mg/kg) to inhibit GPVI signaling, or dabigatran etexilate (chow containing 10 mg/g) or recombinant hirudin (50 mg/kg) to inhibit thrombin activity. Thrombosis was assessed in the carotid artery FeCl 3 model. The carotid artery was exposed and 8% FeCl 3 applied for 3 mins. Blood flow was observed for 30 mins and occlusion was defined as no blood flow for 2 mins. To study susceptibility to viral infection, mice were challenged intranasally with a mouse-adapted H1N1 influenza A virus (H1N1 IAV PR8; 0.02 hemagglutination assay units), which induces mortality in 20% of WT mice. Mortality was defined as body weight loss greater than 25%, which required euthanasia. Results: As expected, PAR4 fl/fl;PF4Cre + platelets were unresponsive to thrombin or PAR4-specific stimulation, while the response to other agonists was retained. In the saphenous vein laser injury hemostasis model, PAR4 fl/fl;PF4Cre + mice were able to rapidly form a hemostatic platelet plug, but the majority of plugs (7/8) were unstable and re-opened after several minutes, leading to severely prolonged total bleeding times. We observed similar findings in global PAR4 -/- mice with 8/12 plugs re-opening. To investigate the mechanism mediating initial platelet plug formation, we inhibited GPVI signaling in PAR4 fl/fl;PF4Cre + mice using the Btk inhibitor ibrutinib. Ibrutinib administration shortened time to plug re-opening in PAR4 fl/fl;PF4Cre + mice but plugs were still able to form, which is likely mediated by GPIbα/VWF. We observed a similar phenotype to PAR4 fl/fl;PF4Cre + mice in mice treated with the direct thrombin inhibitor hirudin, suggesting thrombin is the primary activator of PAR4 during hemostatic plug formation. In the FeCl 3-induced carotid artery thrombosis model, both PAR4 fl/fl;PF4Cre + and PAR4 -/- mice were significantly protected compared to controls. Finally, when challenged with the mouse-adapted H1N1 IAV PR8, PAR4 fl/fl;PF4Cre + mice demonstrated similar body weight loss and survival as littermate controls. Conclusions: Our results in mice demonstrate that 1) platelet PAR4 is not required for initial hemostatic plug formation but is necessary for maintaining hemostatic plug stability, 2) loss of platelet PAR4 protects from arterial thrombosis, and 3) platelet PAR4 does not alter the course of H1N1 IAV infection, at least at the virus dose used in this study. In summary, we generated a novel mouse line carrying a floxed PAR4 allele which can be used to investigate cell-specific roles of PAR4 in disease. Disclosures No relevant conflicts of interest to declare.

Protection of Human Factor V from Activated Protein C Using a Monoclonal Antibody-Biochemical Characterization and Evaluation Using a Mouse Injury Model

Hemophilia is an X-linked bleeding disorder resulting in deficiency of FVIII (hemophilia A; HA) or FIX (HB). Despite treatment with infused FVIII or FIX, people with hemophilia still experience joint bleeding and impaired quality of life. Furthermore, the development of neutralizing antibodies to FVIII or FIX remains a major complication of therapy. To this end, several innovative therapeutic advances which rebalance the coagulation system by targeting anticoagulant pathways (antithrombin, tissue factor pathway inhibitor, and activated protein C (APC)) are particularly interesting as they should be effective for both HA and HB, with and without inhibitors, and may also be useful to treat certain rare bleeding disorders. Many of these approaches are in clinical trials and show promise. However, each of these anticoagulant pathways target multiple coagulation factors, and APC plays an important cellular signaling role. In the current study, we developed a monoclonal antibody (GB5) that targets human FV/FVa which could serve as an alternative therapeutic approach to enhance thrombin generation and prevent bleeding in different clinical situations. Monoclonal antibody GB5 was identified following screening of hybridoma clones derived from mice immunized with human FV. The initial screen identified GB5 as an antibody that could promote thrombin generation (TG) in the presence of APC. To characterize the antibody, purified GB5 was attached to a biosensor tip and evaluated using biolayer interferometry. Analysis of biosensor tracings revealed GB5 bound specifically and with high affinity to both FV (K d = 0.6 nM; 2 experiments) and FVa (K d = 0.7 nM; 2 experiments). A TG assay with pooled normal human plasma (NHP) or HA plasma in the absence or presence of APC was used to assess the functional effects of GB5. In the absence of APC, GB5 had a minimal effect on the TG profile initiated with low tissue factor (0.2 pM). As expected, the addition of APC (2 nM, final) to NHP or HA plasma substantially reduced peak thrombin. However, GB5 dose-dependently increased peak thrombin in the presence of APC in NHP or HA plasma and restored TG to normal levels at ~20 nM. GB5 also protected FV from APC when assessed using a purified prothrombin activation assay. Western blotting experiments using FV proteolyzed with thrombin, APC, or plasmin revealed that GB5 binds the light chain A3-C1-C2. Preliminary experiments revealed that GB5 does not cross react with mouse FV. To evaluate the effect of GB5 in vivo, wild-type (WT) mice were treated with a liver targeted antisense oligonucleotide (ASO) to knockdown mouse plasma FV (ASO-FV; 40 mg/kg; FV-ASO mice). Mouse platelet FV was not impacted. To supplement the plasma compartment with FV, the FV-ASO mice were administered human FV (hFV-mice). This experimental set-up allowed for clot formation to largely be dependent on hFV using the mouse laser injury model. In these studies, we compared the kinetics of thrombus formation in WT mice, FV-ASO mice, or hFV-mice (n=2-3/group; 7-10 thrombi per group) for 10-15 minutes following laser injury. We found that platelet and fibrin accumulation was robust in WT mice and almost undetectable in FV-ASO mice (consistent with the lack of plasma FV), while clot formation was low but detectable in hFV-mice. There was no obvious difference in thrombus size when GB5 was given to WT or FV-ASO mice. However, in hFV-mice (200 μg/kg hFV), GB5 increased platelet and fibrin accumulation to levels seen with WT mice. Quantitative analysis revealed that compared to hFV mice alone, GB5 increased platelet accumulation 3-fold and fibrin (3-6-fold accumulation. Significantly greater accumulation of platelets and fibrin (~30-fold) was observed when higher amounts of hFV (400 μg/kg) were co-infused with GB5 compared to hFV-mice without antibody. Together, these results demonstrate that monoclonal antibody GB5 binds to hFV/FVa with high affinity and confers APC resistance. Using a unique mouse model to assess human FV, we found that GB5 enhanced clot formation in vivo using the laser injury model. From a mechanistic standpoint, these data show that protecting FV from APC translates to greater thrombin generation in vivo and suggest this approach may be useful to treat bleeding disorders such as HA and HB. Additional injury models using the hFV-mice and hemophilic mice to assess the effectiveness of GB5 are ongoing. Disclosures Crosby: Ionis Pharmaceuticals: Other: Jeffrey is a current employee of Ionis Pharmaceuticals. Revenko: Ionis Pharmaceuticals: Other: Alexey is a current employee of the company.. MacLeod: Ionis Pharmaceuticals: Other: Robert is a current employee of the company. Monia: Ionis Pharmaceuticals: Other: Brett is a current employee of the company. .

Safety During Ureteroscopy: Radiation, Eyes, and Ergonomics

It is known that urologic surgeons are at risk of work-place injury due to the physical requirements of operating and exposure to hazards. These hazards include radiation, exposure to body fluids, use of laser energy, and orthopedic injury due to the physical nature of operating. The risks that these hazards present can be mitigated by implementing several evidence-based safety measures. The methods to protect against radiation exposure include keeping radiation usage in the operating room as low as reasonably achievable, donning lead aprons, and wearing protective glasses. Additionally, protective glasses decrease the risk of eye injury from laser injury and exposure to body fluids. Finally, practicing sound surgical ergonomics is essential to minimize the risk of orthopedic injury and promote career longevity. The interventions discussed herein are simple and easy to implement in one's daily practice of urology.

Macrophage-derived interleukin-6 is necessary and sufficient for choroidal angiogenesis

Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Interleukin-6 (IL6) is a pro-inflammatory and pro-angiogenic cytokine that is correlated with AMD progression and nAMD activity. We hypothesize that anti-IL6 therapy is a potential nAMD therapeutic. We found that IL6 levels were increased after laser injury and expressed by macrophages. Il6-deficiency decreased laser-induced CNV area and exogenous IL6 addition increased choroidal sprouting angiogenesis. Il6-null mice demonstrated equally increased macrophage numbers as wildtype mice. At steady state, IL6R expression was detected on peripheral blood and ocular monocytes. After laser injury, the number of IL6R+Ly6C+ monocytes in blood and IL6R+ macrophages in the eye were increased. In human choroid, macrophages expressed IL6, IL6R, and IL6ST. Furthermore, IL6R+ macrophages displayed a transcriptional profile consistent with STAT3 (signal transducer and activator of transcription 3) activation and angiogenesis. Our data show that IL6 is both necessary and sufficient for choroidal angiogenesis. Macrophage-derived IL6 may stimulate choroidal angiogenesis via classical activation of IL6R+ macrophages, which then stimulate angiogenesis. Targeting IL6 or the IL6R could be an effective adjunctive therapy for treatment-resistant nAMD patients.

Dendritic cells play no significant role in the laser-induced choroidal neovascularization model

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3−/− and Flt3l−/− mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3−/− mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3−/− mice. In Flt3l−/− mice, each DC subsets was severely reduced after laser injury. Neither Flt3−/− or Flt3l−/− mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.

Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

Macrophages stimulate epicardial VEGFaa to trigger cardiomyocyte proliferation in larval zebrafish heart regeneration

Cardiac injury induces a sustained innate immune response in both zebrafish and mammals. Macrophages, highly plastic immune cells, perform a range of both beneficial and detrimental functions during mammalian cardiac repair yet their precise roles in zebrafish cardiac regeneration are not fully understood. Here we characterise cardiac regeneration in the rapidly regenerating larval zebrafish laser injury model and use macrophage ablation and macrophage-less irf8 mutants to define the requirement of macrophages for key stages of regeneration. We found macrophages to display cellular heterogeneity and plasticity in larval heart injury as in mammals. Live heartbeat-synchronised imaging and RNAseq revealed an early proinflammatory macrophage phase which then resolves to an anti-inflammatory, profibrotic phase. Macrophages are required for cardiomyocyte proliferation but not for functional or structural recovery following injury. Macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium which upregulates mitogen VEGFaa. Experimental perturbation of VEGF signalling confirmed VEGFaa to be an important inducer of cardiomyocyte proliferation revealing a previously unrecognised mechanism by which macrophages aid cardiac regeneration.

Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

A chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.