Hyaloid Vasculature as a Major Source of STAT3 + (Signal Transducer and Activator of Transcription 3) Myeloid Cells for Pathogenic Retinal Neovascularization in Oxygen-Induced Retinopathy

Objective: Myeloid cells are critically involved in inflammation-induced angiogenesis, although their pathogenic role in the ischemic retina remains controversial. We hypothesize that myeloid cells contribute to pathogenic neovascularization in retinopathy of prematurity through STAT3 (signal transducer and activator of transcription 3) activation. Approach and Results: Using the mouse model of oxygen-induced retinopathy, we show that myeloid cells (CD45 + IsolectinB4 [IB4] + ) and particularly M2-type macrophages (CD45 + Arg1 + ), comprise a major source of STAT3 activation (pSTAT3) in the immature ischemic retina. Most of the pSTAT3-expressing myeloid cells concentrated at the hyaloid vasculature and their numbers were strongly correlated with the severity of pathogenic neovascular tuft formation. Pharmacological inhibition of STAT3 reduced the load of IB4 + cells in the hyaloid vasculature and significantly reduced the formation of pathogenic neovascular tufts during oxygen-induced retinopathy, leading to improved long-term visual outcomes (ie, increased retinal thickness and scotopic b-wave electroretinogram responses). Genetic deletion of SOCS3 (suppressor of cytokine signaling 3), an endogenous inhibitor of STAT3, in myeloid cells, enhanced pathological and physiological neovascularization in oxygen-induced retinopathy, indicating that myeloid-STAT3 signaling is crucial for retinal angiogenesis. Conclusions: Circulating myeloid cells may migrate to the immature ischemic retina through the hyaloid vasculature and contribute to retinal neovascularization via activation of STAT3. Understanding how STAT3 modulates myeloid cells for vascular repair/pathology may provide novel therapeutic options in pathogenic angiogenesis.

Neonatal and Juvenile Ocular Development in Göttingen Minipigs and Domestic Pigs: A Histomorphological and Immunohistochemical Study

Pigs are considered one of the relevant animal models for ocular research as they share several histological and anatomical similarities with the human eye. With the increasing interest in juvenile animal models, this study aimed to describe the postnatal development of ocular structures in 16 Göttingen minipigs and 25 F2 domestic pigs, between birth and 6 months of age, using histopathology and immunohistochemistry against Ki-67, caspase-3, calbindin, glial fibrillary acidic protein, rhodopsin, and synaptophysin. All ocular structures in both pig breeds were incompletely developed at birth and for variable periods postnatally. Noteworthy histological features of immaturity included vascularization in the corneal stroma in neonatal Göttingen minipigs, increased cellularity in different substructures, remnants of the hyaloid vasculature, short and poorly ramified ciliary body processes, and a poorly developed cone inner segment. Increased cellular proliferation, highlighted by abundant Ki-67 immunolabeling, was observed in almost all developing structures of the pig eye for variable periods postnatally. Apoptosis, highlighted with caspase-3 immunolabeling, was observed in the retinal inner nuclear layer at birth and in the regressing hyaloid vasculature remnants. Immunohistochemistry against rhodopsin, synaptophysin, and calbindin demonstrated the short size of the developing photoreceptors and the immature cone inner segment morphology. Calbindin labeling revealed significant differences in the amount of positively labeled cone nuclei between the retinal area centralis and the non–area centralis regions. The elongation of Müller cell processes in the developing retina was shown with glial fibrillary acidic protein. In both pig breeds, the eyes reached histomorphological and immunohistochemical maturity at 6 months of age.

Primary cilia are present on endothelial cells of the hyaloid vasculature but are not required for the development of the blood-retinal barrier

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.

Pax2a regulates angiogenesis to facilitate mmp2-dependent basement membrane remodeling of the optic fissure

ABSTRACTVertebrate retinal development requires timely and precise fusion of the optic fissure (OF). Recent studies have suggested hyaloid vasculature to be involved in optic fissure fusion. In order to examine this link, we analyzed OF fusion and hyaloid vasculogenesis in the zebrafish pax2anoi mutant line. We determined that OF basement membrane (BM) remodeling, normally preceded by F-actin accumulation is mis-regulated in pax2a−/− embryos. Comparing transcriptomic profiles of pax2a−/− and wildtype eyes we discovered a novel connection between regulation of angiogenesis and fusion. Pax2a−/− eyes exhibited a significant reduction of talin1 expression, a regulator of hyaloid vasculature, in addition to increased expression of an anti-angiogenic protease, adamts1. Using 3D and live imaging we observed reduced OF hyaloid vascularization in pax2a−/− embryos. Additionally, pharmacological inhibition of VEGF signaling or adamts1 mRNA overexpression phenocopied the pax2a−/− vasculature, F-actin and BM remodeling phenotypes. Finally, we show that hyaloid vasculature expresses mmp2 which is necessary for remodeling the fissure BM. Taken together we propose a pax2a driven mechanism that restricts anti-angiogenic activity of adamts1 enabling hyaloid vasculature invasion of the OF and delivery of the BM remodeler mmp2.

Functional OCT angiography reveals early physiological dysfunction of hyaloid vasculature in developing mouse eye

Hyaloid vascular system (HVS) is a transient capillary network nourishing developing eye. Better study of the HVS regression correlated with eye development is essential for in-depth understanding of the nature of vision system. In this study, we demonstrate the feasibility of longitudinal optical coherence tomography (OCT) and OCT angiography (OCTA) monitoring of the HVS in C57BL/6J mice. OCT enables morphological monitoring of the HVS regression, and OCTA allows physiological assessment of the HVS involution correlated with eye development. Functional OCTA reveals early physiological dysfunction before morphological regression of the hyaloid vasculature in developing mouse eye. We anticipate that noninvasive, simultaneous OCT/OCTA observation of morphological regression and physiological degradation in normal and diseased animal models will be valuable to unravel the complex mechanisms of the HVS regression correlated with normal eye development and abnormal persistent hyaloid conditions. Impact statement Hyaloid vascular system (HVS) is known to have an essential role in the eye development. However, established knowledge of the HVS largely relies on end-point studies with biochemically fixed tissues, lacking a full description of the natural dynamics of the HVS correlated with eye development. An imaging methodology for noninvasive, longitudinal, and high-resolution monitoring of the HVS is important not only for better understanding of the nature of the vision system and is also valuable for better study of abnormal eye conditions. Here, we report the feasibility of in vivo optical coherence tomography (OCT) and OCT angiography (OCTA) imaging of the HVS regression in developing mouse eye. OCT enables morphological imaging of the HVS structure, and OCTA allows functional assessment of the HVS physiology correlated with eye development.