Histochemical characteristics of regressing vessels in the hyaloid vascular system of neonatal mice: Novel implication for vascular atrophy

Abstract

The hyaloid vasculature constitutes a transitory system nourishing the internal structures of the developing eye, but the mechanism of vascular regression and its cell biological characteristics are not fully understood. The present study aimed to reveal the specificity of the hyaloid vessels by a systematic immunohistochemical approach for marker substances of myeloid cells and the extracellular matrix (ECM) in neonatal mice. Macrophages immunoreactive for F4/80, cathepsin D, and LYVE-1 gathered around the vasa hyaloidea propria (VHP), while small round cells in vascular lumen of VHP were selectively immunoreactive for galectin-3; their segmented nuclei and immunoreactivities for Ly-6G, CD11b, and myeloperoxidase indicated their neutrophilic origin. VHP possessed thick ECM and a dense pericyte envelope as demonstrated by immunostaining for laminin, type IV collagen, integrin β1, and NG2. The galectin-3 cells loosely aggregated with numerous erythrocytes in the lumen of hyaloid vessels in a manner reminiscent of vascular congestion. Galectin-3 is known to polymerize and form a complex with ECM and NG2 as well as recruit leukocytes on the endothelium. Observation of galectin-3 KO mice implicated the involvement of galectin-3 in the regression of hyaloid vasculature. Since macrophages may play central roles including blocking of the blood flow and the induction of apoptosis in the regression, galectin-3 neutrophils may play a supportive role in the macrophage-mediated involution of the hyaloid vascular system.

Bush-like integrin filament networks associated with hyaloid vasculature in murine neonate eyes

“…The present immunostaining for integrin β 1 demonstrated the positive labeling of macrophages with dotted appearances in the cytoplasm, parallel to the fragmentation and disappearance of integrin bushes. This image suggests the simple uptaking and degradation of integrin by associated macrophages with a higher phagocytotic activity than shown in our previous study (11). However, there is a possibility that endothelial cells themselves uptake and degrade integrin with the aid of macrophages.…”

“…However, the integrin-immunoreactive round cells almost disappeared after birth, while the networks of integrin fibers developed more extensively. Immunostaining of the neonatal ocular tissues at postnatal days 1-3 using CD31 antibody and laminin antibody revealed the entire images of the VHP (11). The VHP possessed a thick basement membrane consisting of extracellular matrix elements including laminin and β 1 integrin.…”

“…The vitreous humour is essentially composed of the extracellular matrix, rich in hyaluronan. Macrophages, densely distributed along the VHP, expressed the LYVE-1 hyaluronan receptor (11,20). This is helpful for the residing of macrophages in the hyaluronan-rich vitreous.…”

“…Using macrophage-deficient mice, it has been shown that macrophages play an essential role in the regression of the hyaloid vascular system (12). Recently, we reported the involvement of galectin-3-expressing granulocytes in the regression process (11). Another characteristic of the VHP is to float in the vitreous, which lacks any firm structural components.…”

Assessment and Characterization of Hyaloid Vessels in Mice

“…Limitations of this isolation protocol also exist, including the complexity of performing the fine dissection and the difficulty of long-term preservation of samples due to the nature of fragile tissue. Nevertheless, isolating and flat mounting hyaloid vessels is still the most comprehensive and intuitive way to study hyaloid vasculature, to promote ocular and angiogenesis studies 10,17,27,[29][30][31] .…”