Anti‐angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor‐A (VEGF‐A) have revolutionized treatment of retinal vascular diseases including age‐related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti‐VEGF‐A monotherapy efficacy by targeting both VEGF‐A and angiopoietin‐2 (ANG‐2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF‐A. Simultaneous VEGF‐A and ANG‐2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain‐exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF‐A and ANG‐2. RG7716 showed greater efficacy than anti‐VEGF‐A alone in a non‐human primate laser‐induced CNV model after intravitreal delivery. Modification of RG7716's FcRn and FcγR binding sites disabled the antibodies' Fc‐mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential next‐generation therapy for neovascular indications of the eye.

Diabetic retinopathy (DR) is a retinal microvascular disease characterized by inflammatory and angiogenic pathways. In this study, we evaluated NLRP3 inflammasome in a double transgenic mouse model, Akimba (Ins2AkitaxVEGF+/−), which demonstrates hyperglycemia, vascular hyperpermeability and neovascularization seen in the proliferative DR. Retinal structural integrity, vascular leakage and function were examined by fundus photography, fluorescein angiography, optical coherence tomography, retinal flat mounts, laser speckle flowgraphy (LSFG), and electroretinography in Akimba and its parental strains, Akita (Ins2Akita) and Kimba (trVEGF029) mice. Inflammatory mechanisms involving NLRP3 inflammasome were investigated using real time-PCR, immunohistochemistry, ELISA and western blots. We observed an increased vascular leakage, reduced retinal thickness, and function in Akimba retina. Also, Akimba retina depicts decreased relative flow volume measured by LSFG. Most importantly, high levels of IL-1β along with increased NLRP3, ASC, and Caspase-1 at mRNA and protein levels were observed in Akimba retina. However, the in vivo functional role remains undefined. In conclusion, increased activation of macroglia (GFAP), microglia (Iba-1 and OX-42) and perivascular macrophages (F4/80 and CD14) together with pro-inflammatory (IL-1β and IL-6) and pro-angiogenic markers (PECAM-1, ICAM-1, VEGF, Flt-1, and Flk-1), suggested a critical role for NLRP3 inflammasome in the Akimba mouse model depicting advanced stages of DR pathogenesis.

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SummaryBlood flow regulation in pancreatic islets is critical for function but poorly understood. Here, we establish an in vivo imaging platform in a non-human primate where islets transplanted autologously into the anterior chamber of the eye are monitored non-invasively and longitudinally at single-cell resolution. Engrafted islets were vascularized and innervated and maintained the cytoarchitecture of in situ islets in the pancreas. Blood flow velocity in the engrafted islets was not affected by increasing blood glucose levels and/or the GLP-1R agonist liraglutide. However, islet blood flow was dynamic in nature and fluctuated in various capillaries. This was associated with vasoconstriction events resembling a sphincter-like action, most likely regulated by adrenergic signaling. These observations suggest a mechanism in primate islets that diverts blood flow to cell regions with higher metabolic demand. The described imaging technology applied in non-human primate islets may contribute to a better understanding of human islet pathophysiology.