Loss of <i>Lrp5</i> causes retinal hypovascularization and neovascularization.

<p>(A) ColIV whole mount IF staining showing retinal vessels of <i>Lrp5</i>^<i>-/-</i> and control mice at P9 and P30. Quantification of vascular sprout numbers at P5 shown at right. (B) Adult <i>Lrp5</i>^<i>-/-</i> retinas showing persistent hyaloid vessels (black arrows, CD31 IHC staining), aneurysms (open arrow, CD31 IHC staining), neovascular overgrowth (white arrows, fibronectin IF staining) and lack of IPL and OPL vascular development (lower panels, green: FITC-Dextran perfusion). (C) FITC-Dextran perfusion (green) showing retinal vascular leakage (white arrows) in adult <i>Lrp5</i>^<i>-/-</i> mice compared to control. Scale bars = 100μm. (D) EM analysis of endothelium of <i>Lrp5</i>^<i>-/-</i> and control retinas at P5, P8 and P30. Arrows point to area of endothelial fenestration. Scale bars = 500nm. (E) Total amounts of VEGF protein in retinas of <i>Lrp5</i>^<i>-/-</i> and control mice at P5, P8 and P30. Each ELISA was done in duplicate and normalized to total retinal protein amount. <i>n</i> = 9, 5, 12 for controls (at P5, P8, P30) and 9, 8, 8 for <i>Lrp5</i>^<i>-/-</i> (at P5, P8, P30). * <i>p</i><0.05, ** <i>p</i><0.01. Data are represented as means ± SD. Ctrl, control.</p

Endothelium-derived <i>Lrp6</i> is dispensable for retinal vascular development.

<p>Whole mount IF staining of ColIV (red) and FITC-Dextran perfusion showing (A) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/+</i><i>;Lrp6</i>^<i>fl/+</i> CKO mice. (B) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/+</i><i>;Lrp6</i>^<i>fl/fl</i> CKO mice. (C) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i> CKO mice. (D) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i><i>;Lrp6</i>^<i>fl/+</i> CKO mice. All mice are 8 weeks of age. Scale bars = 100nm.</p

Retinal vascular phenotype in mice with conditional knockout of <i>Lrp5</i> in myeloid/microglial cells.

<p>(A) Whole mount IF staining with IB4 (blue) or ColIV antibody (right panel, green) showing distribution of <i>LysM-Cre;tdTomato</i>⁺ (red) myeloid and microglial cells in retinas at P5, P8 and 6w. Most <i>LysM-Cre;tdTomato</i>⁺ myeloid cells were also F4/80⁺ (green) (white rectangle in left panel indicates area shown at higher magnification in the upper right corner inset). (B) Localization of <i>LysM-Cre;tdTomato</i>⁺ myeloid cells in three adult (6w) retinal vascular beds. Green: ColIV. (C) Retinal vasculature of <i>LysM-Cre;Lrp5</i>^<i>fl/-</i> CKO mice (12w). Red: IB4. (D) Distribution of <i>CD11b-Cre;tdTomato</i>⁺ (red) myeloid and microglial cells in retinas at P6 (left and mid panels, green: IB4) and 4w (right panel, cross section). Blue: Hoechst. (E) Localization of <i>CD11b-Cre;tdTomato</i>⁺ signals (red) in myeloid, microglial, Müller glial and perivascular macrophage cells in adult (4w) retina. Blue: IB4. White rectangle in right panel outlines cell shown at higher magnification in upper right corner inset. (F) Retinal vasculature of <i>CD11b-Cre;Lrp5</i>^<i>fl/-</i> CKO mice (4w). Green: FITC-Dextran perfusion. Scale bars = 100nm.</p

Conditionally restoring <i>Lrp5</i> in endothelial but not myeloid cells rescues retinal vascular defects in <i>Lrp5</i>^<i>-/-</i> mice.

<p>(A) FITC-Dextran perfusion showing distorted NFL and IPL vessels and lack of OPL vascular development in <i>Lrp5</i>^{<i>a214v(n)/-</i>} hypomorph mice (lower panels) compared to controls (upper panels) both in retinal whole mount (4w) and cross section (2m) images. (B) <i>VE-Cad-Cre;Lrp5</i>^{<i>a214v(n)/+</i>} (upper left panels, FITC-Dextran perfusion), <i>VE-Cad-Cre;Lrp5</i>^{<i>a214v(n)/-</i>} (lower left panels, FITC-Dextran perfusion) and <i>Flk1-Cre;Lrp5</i>^{<i>a214v(n)/-</i>} (upper right panels, red: ColIV, green: IB4) mice all developed a normalized three-tier retinal vascular structure, while <i>LysM-Cre;Lrp5</i>^{<i>a214v(n)/-</i>} (lower right panels, red: ColIV) mice displayed similar retinal vascular abnormalities compared to control <i>Lrp5</i>^{<i>a214v(n)/-</i>} mice (A, lower panels). Quantification of vascular branch points in OPL is shown in graph at right; **P<0.01, ns not significant. All mice are between 4 to 5 weeks of age except otherwise labeled. Scale bars = 100nm.</p

Conditional knockout of <i>Lrp5</i> with <i>Tie2-Cre</i> but not <i>VE-Cad-Cre</i> recapitulates retinal vascular defects in <i>Lrp5</i>^<i>-/-</i> mice.

<p>(A and B) ColIV IF staining (red) and FITC-Dextran perfusion (green) showing adult retinal vasculature in <i>VE-Cad-Cre;Lrp5</i>^<i>fl/-</i> and <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i> CKO mice (4w) compared to <i>Lrp5</i>^<i>fl/fl</i> control. Arrows: hyaloid vessels; open arrows: neovascular tufts; arrowheads: neovascular overgrowth. At right: Quantification of vascular branch points in OPL at 4w; ns not significant. (C) TdTomato signals in <i>VE-Cad-Cre;tdTomato</i> (8w) and <i>Tie2-Cre;tdTomato</i> (7w) mice showing predominant endothelial expression of <i>VE-Cad-Cre and Tie2-Cre</i> in the NFL, IPL and OPL of the retina. Note that <i>VE-Cad-Cre</i> is also widely expressed in myeloid cells around the vessels in the NFL (upper left panel, also see D). Arrows point to IB4 (green) positive vascular area with negative <i>VE-Cad-Cre;tdTomato</i> signals in a P5 mouse indicating incomplete <i>VE-Cad-Cre</i> recombination. (D) Myeloid and microglial localization of <i>VE-Cad-Cre;tdTomato</i> and <i>Tie2-Cre;tdTomato</i> signals in P5 (left and mid-left panels) and adult (right and mid-right panels) retinas. Arrows point to myeloid cells and arrowheads point to microglial cell. Green: F4/80 IF staining. Scale bars = 100nm.</p

<i>Flk1-Cre</i>^{<i>Breier</i>} is specifically expressed in endothelial cells and conditional knockout of <i>Lrp5</i> with <i>Flk1-Cre</i>^{<i>Breier</i>} recapitulates retinal vascular defects in <i>Lrp5</i>^<i>-/-</i> mice.

<p>(A) Specific endothelial location of <i>Flk1-Cre</i>^{<i>Breier</i>}<i>;tdTomato</i> signals in developing retinas (P5, upper panels) overlapping with IB4 IF signals (blue). F4/80 IF staining (green) showing that most macrophages were tdTomato negative. In adult retina, <i>Flk1-Cre</i>^{<i>Breier</i>} was specifically expressed in all three layers of the retinal vascular beds (lower panels) with no signs of any myeloid cell expression. Penetrance of <i>Flk1-Cre</i>^{<i>Breier</i>} expression in ECs could also be incomplete as shown in (D). (B) Specific endothelial location of <i>Flk1-Cre</i>^{<i>Breier</i>}<i>;tdTomato</i> signals in adult (4w) bone marrow, cortical bone and skeletal muscle. M: muscle; CB: cortical bone; BM: bone marrow. Blue: Hoechst. (C) Whole mount IF staining of ColIV (red) and IB4 (green) showing retinal vasculature in adult (4w) <i>Flk1-Cre</i>^{<i>Breier</i>}<i>;Lrp5</i>^<i>fl/-</i> CKO mice with disorganized NFL vessels, vertical vessel branches terminating in ball-like structures in the IPL and lack of OPL vascular bed. Arrow points to persistent hyaloid vessels. Note that regions with vascular abnormalities often had patchy normal-looking areas located in the neighborhood. In the selected OPL image, an area with well-developed vessels is next to another with no vascular development. (D) Whole mount IF staining of IB4 (green) showing that normally developed vascular areas in <i>Flk1-Cre;Lrp5</i>^<i>fl/-</i><i>;tdTomato</i> retinas (8w) included many <i>Flk1-Cre;</i>tdTomato negative vessels (arrows), whereas abnormal vessel structures were all tdTomato positive (open arrows). Scale bars = 100μm.</p

The increase in bone mineral density by bisphosphonate with active vitamin D analog is associated with the serum calcium level within the reference interval in postmenopausal osteoporosis

<p><b>Objectives:</b> To examine the factors associated with increase in lumbar spine bone mineral density (LS-BMD) by bisphosphonates (BPs) with active vitamin D analog (aVD).</p> <p><b>Methods:</b> Two independent postmenopausal osteoporotic patients treated by BPs with aVD for 24 months (Study 1: <i>n</i> = 93, Study 2: <i>n</i> = 99) were retrospectively analyzed.</p> <p><b>Results:</b> In Study 1, LS-BMD of the patients significantly increased for 24 m (5.4%, <i>p</i> < .001). A multiple regression analysis among baseline characteristics revealed that serum calcium (sCa: 8.5–10.5 mg/dL) was associated with an increased LS-BMD by treatment (<i>r</i>²: 0.088, <i>p</i> = .02). While average sCa of the patients was 9.2 mg/dL before treatment, it increased time-dependently to 9.6 mg/dL for 24 m by treatment. As each patient had their LS-BMD five times during the study, there were four instances of %LS-BMD in each patient, resulting in 372 instances of %LS-BMD in Study 1. The smallest Akaike’s information criterion value for the most appropriate cut-off levels of sCa for %LS-BMD by treatment every 6 m was 9.3 mg/dL. The %LS-BMD by treatment for 6 m during 24 m period in patients with sCa ≥9.3 mg/dL (1.5%) was significantly higher than that in patients with sCa <9.3 mg/dL (0.8%, <i>p</i> = .038). The results of Study 2 were similar to those of Study 1, confirming the phenomena observed.</p> <p><b>Conclusion:</b> sCa was associated with an increased LS-BMD by BPs with aVD.</p