Bim expression in endothelial cells and pericytes is essential for regression of the fetal ocular vasculature.

Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision

Bcl-2 expression in pericytes and astrocytes impacts vascular development and homeostasis.

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-

Increased EC number in retinas from conditional Bim mice.

<p>Retinas from P21 and P42 mice were prepared by trypsin digest and HE/PAS staining. EC and PC were then quantitated per x400 field of view. Scale bar = 100 μm. Experiments were repeated with eyes from >10 mice with similar results. Four quadrants per eye were counted for the quantitative assessment of this data is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178198#pone.0178198.t001" target="_blank">Table 1</a>.</p

Bim expression increases during hyperoxia.

<p>Wild-type mice were subjected to OIR at P7. RNA was prepared from the retina of wild-type mice at the noted times. Bim expression was analyzed by qPCR. Please note increased Bim expression with hyperoxia (P12). Samples were done in triplicate and repeated twice. (n = 6, ***P<0.001).</p

Decreased proliferation in retinas from conditional Bim mice.

<p>Proliferating cells in P14 wild-type, Bim^EC and Bim^PC mouse retinas were assessed by anti-Ki-67 staining (red) and co-stained with Isolectin-B4-FITC (green) to visualize the vasculature. The data in each bar are the mean number of Ki-67 positive cells counted in each retina. Please note that the number of proliferating cells is lower in retinas from conditional Bim mice compared to their wild-type counterparts (n = 6, ****P< 0.0001). Scale bar equals 400 μm.</p

Retinal vascular cell numbers.

<p>Retinal vascular cell numbers.</p

Enhanced deep vascular plexus formation in Bim^EC mice.

<p>In <b>Panel A</b>, retinas from P10 wild-type, Bim^EC and Bim^PC mice were wholemount stained with Isolectin B4 and the superficial layer imaged (25x). In <b>Panel B</b> the mean number of vertical sprouts were quantified in each retina. (n = 5, *P<0.05). In <b>Panel C</b>, the area covered by the vertical sprouts for each retina was quantified. (n = 5, **P<0.01)</p

P21 Tomato^EC and Tomato^PC mice were perfused with FITC-wheat germ agglutinin to visualize the vasculature (green) with VE-cadherin-cre expressing cells (endothelial cells; red) in the upper panel and Pdgfrb-cre expressing cells in the lower panel at x200 with a higher magnification (Tomato) shown at x400.

<p>P21 Tomato^EC and Tomato^PC mice were perfused with FITC-wheat germ agglutinin to visualize the vasculature (green) with VE-cadherin-cre expressing cells (endothelial cells; red) in the upper panel and Pdgfrb-cre expressing cells in the lower panel at x200 with a higher magnification (Tomato) shown at x400.</p

Hyaloid regression is regulated by Bim expression in vascular cells.

<p><b>Panel A</b> demonstrates DAPI stained hyaloid vessel preparations from postnatal day 10 wild-type and conditional Bim knockout mice. Please note lack of Bim expression in EC or PC attenuated hyaloid vessel regression. (n =, ****P<0.0001) Scale bar equals 2000 μm. In <b>Panel B</b>, a representative image is shown of hyaloid vessels from 10 week old mice imaged using a Micron III indirect camera. Fundus images were taken prior to an intraperitoneal injection of 50 μl sodium fluorescein. While the retina was in focus on the Micron III, images were taken as the hyaloid vessels filled with fluorescein. These studies were performed by imaging hyaloid vessels from 5 mice (n = 5) with similar results.</p

Lack of EC or PC Bim expression does not attenuate hyperoxia-driven vessel obliteration.

<p>Quantitative assessment of vessel obliteration and neovascularization in mice previously exposed to a cycle of hyperoxia and room air (OIR). Retinas from P12 and P17 Bim^Flox/Flox (WT) and conditional Bim littermates were wholemount stained with anti-collagen IV to visualize the vasculature (Panels A,C). The area of vessel obliteration relative to the whole retina was quantitated at P12 (n = 5, P>0.05, Panel B) and the non-perfused area remaining relative to the whole retina at P17 was quantitated (Panel D; n = 5, **P<0.01, ***P<0.001). Panel E is a quantitative assessment of the neovascularization (n = 5, P> 0.05). For Panel A magnification is at 2.5 X while Panel C is at 1.25 X.</p