Susumu Sakimoto

Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels

Vasculogenesis, the in‐situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem‐like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony‐forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de‐novo long‐term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub‐population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem‐like ECs will lead to the identification of new targets for vascular regeneration therapy.

microRNA-125b inhibits tube formation of blood vessels through translational suppression of VE-cadherin

Angiogenesis is controlled positively or negatively by extrinsic and intrinsic molecular cues in endothelial cells (ECs); in the tumor microenvironment, the action of positive regulators exceeds that of negative regulators. Thus, overinduction of negative regulators may inhibit tumor angiogenesis. MicroRNAs (miRNAs or miRs) are endogenous short noncoding RNAs regulating gene expression either through translational inhibition or destabilization of target mRNA. Here, we show that miR-125b expression is transiently induced in ECs on stimulation with vascular endothelial growth factor or by ischemia. miR-125b inhibits translation of vascular endothelial (VE)-cadherin mRNA and in vitro tube formation by ECs. Injection of miR-125b into the tumor inhibited VE-cadherin expression by ECs and induced nonfunctional blood vessel formation, resulting in inhibition of tumor growth. It has been suggested that pro-angiogenic signals in ECs also upregulate anti-angiogenic molecules simultaneously via negative feedback. Because miR-125b induction in ECs is transient after pro-angiogenic stimulation, prolonged overexpression of miR-125b could result in blood vessel regression. Thus, miR-125b may be useful in cancer therapy by causing the collapse of the lumen of ECs.

Suppression and regression of choroidal neovascularization in mice by a novel CCR2 antagonist, INCB3344.

Purpose To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV). Methods CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA). Results The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1 −/− mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001). Conclusions INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration.

A role for endothelial cells in promoting the maturation of astrocytes through the apelin/APJ system in mice

Interactions between astrocytes and endothelial cells (ECs) are crucial for retinal vascular formation. Astrocytes induce migration and proliferation of ECs via their production of vascular endothelial growth factor (VEGF) and, conversely, ECs induce maturation of astrocytes possibly by the secretion of leukemia inhibitory factor (LIF). Together with the maturation of astrocytes, this finalizes angiogenesis. Thus far, the mechanisms triggering LIF production in ECs are unclear. Here we show that apelin, a ligand for the endothelial receptor APJ, induces maturation of astrocytes mediated by the production of LIF from ECs. APJ (Aplnr)- and Apln-deficient mice show delayed angiogenesis; however, aberrant overgrowth of endothelial networks with immature astrocyte overgrowth was induced. When ECs were stimulated with apelin, LIF expression was upregulated and intraocular injection of LIF into APJ-deficient mice suppressed EC and astrocyte overgrowth. These data suggest an involvement of apelin/APJ in the maturation process of retinal angiogenesis.

Arf6 regulates tumour angiogenesis and growth through HGF-induced endothelial β1 integrin recycling

Anti-angiogenic drugs targeting vascular endothelial cell growth factor receptor have provided modest clinical benefit, in part, owing to the actions of additional angiogenic factors that stimulate tumour neoangiogenesis in parallel. To overcome this redundancy, approaches targeting these other signalling pathways are required. Here we show, using endothelial cell-targeted mice, that the small GTPase Arf6 is required for hepatocyte growth factor (HGF)-induced tumour neoangiogenesis and growth. Arf6 deletion from endothelial cells abolishes HGF-stimulated β1 integrin recycling. Pharmacological inhibition of the Arf6 guanine nucleotide exchange factor (GEF) Grp1 efficiently suppresses tumour vascularization and growth. Grp1 as well as other Arf6 GEFs, such as GEP100, EFA6B and EFA6D, regulates HGF-stimulated β1 integrin recycling. These findings provide insight into the mechanism of HGF-induced tumour angiogenesis and offer the possibility that targeting the HGF-activated Arf6 signalling pathway may synergize with existing anti-angiogenic drugs to improve clinical outcomes.

Analysis of Retinal Nonperfusion Using Depth-Integrated Optical Coherence Tomography Images in Eyes With Branch Retinal Vein Occlusion

PURPOSE To assess the morphology of areas of complete retinal nonperfusion in eyes with branch retinal vein occlusion (BRVO) by en face images of optical coherence tomography (OCT). METHODS Forty-six eyes with BRVO that underwent swept-source OCT (SS-OCT) and fluorescein angiography were enrolled. Depth-integrated images of the neural retina delineated by automated segmentation algorithm were obtained using SS-OCT. The findings in a 6 × 6-mm area on en face SS-OCT scans at the area of retinal nonperfusion were evaluated. RESULTS Retinal nonperfusion was detected in 25 eyes. Of these, 20 (80%) eyes had multiple concaves of low reflectivity within an area of reticular high reflectivity (honeycomb sign) on depth-integrated images at the area corresponding to retinal nonperfusion. The mean area of retinal nonperfusion and honeycomb sign were 6.72 ± 4.10 mm2 and 4.19 ± 3.37 mm2, respectively. The area of retinal nonperfusion was correlated significantly (r = 0.53, P < 0.001) with the area of the honeycomb sign. The mean retinal thickness in eyes with a honeycomb sign increased significantly (P = 0.017) compared with eyes without a honeycomb sign. Furthermore, after anti-VEGF injection, the mean area of honeycomb sign decreased significantly (P = 0.0013), from 4.23 mm(2) to 0.48 mm(2). CONCLUSIONS Depth-integrated OCT images with automated layer segmentation showed a two-dimensional honeycomb-like structure in retinal nonperfusion with retinal edema. In eyes with BRVO, the thickness of retina did not decrease but increased due to retinal cyst in spite of neural retinal thinning.

Laser-Induced Choroidal Neovascularization in Mice Attenuated by Deficiency in the Apelin-APJ System

PURPOSE To investigate the role of the apelin-APJ system in the development of choroidal neovascularization (CNV). METHODS Experimental CNV was induced by laser photocoagulation in wild-type (WT), apelin-deficient (apelin-KO), and apelin receptor (APJ)-deficient (APJ-KO) mice. The gene expression levels of angiogenic or inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction. APJ expression in CNV lesions was examined by immunohistochemistry. The sizes of the CNV lesions in the three mouse models were measured and compared histologically using isolectin B4 staining. Macrophage recruitment was measured by flow cytometric analysis. Proliferation of endothelial cells was determined using the alamar Blue assay. RESULTS Laser photocoagulation significantly increased expression of apelin and APJ in the retina-retinal pigment epithelium (RPE) complex. APJ immunoreactive cells were found in the CNV lesions and colocalized with platelet endothelial cell adhesion molecule-1, an endothelial cell marker. The sizes of the CNV lesions in apelin-KO or APJ-KO mice decreased significantly compared with those in the WT mice. Macrophages in the RPE complex of the apelin-KO mice, in which gene expression of the inflammatory factors was almost equal to that in WT mice, were recruited as a result of laser photocoagulation to the same degree as in WT mice. In addition, apelin small and interfering RNA (siRNA) suppressed proliferation of endothelial cells independently of vascular endothelial growth factor (VEGF) receptor 2 signaling, while VEGF increased expression of apelin and APJ in human umbilical vein endothelial cells. CONCLUSIONS The results suggested that the apelin-APJ system contributes to CNV development partially independent of the VEGF pathway.

Relationship between grades of macular perfusion and foveal thickness in branch retinal vein occlusion

Background To study the relationship between retinal perfusion around the macula and the foveal thickness in branch retinal vein occlusion (BRVO). Methods Seventy-four eyes of 74 consecutive patients with BRVO were enrolled. We developed a new grading system to evaluate the status of retinal perfusion around the macula in three grades: full perfusion area (FPA), partial perfusion area (PPA), and nonperfusion area (NPA), using fluorescein angiography. The correlation was assessed between these grades and the central foveal thickness (CFT) measured by optical coherence tomography. We also determined the area with the closest correlation between the perfusion status and the foveal thickness by comparing the correlation coefficient in areas of 1-, 2-, and 3-disc diameter (DD) horizontal hemicircles centered at the fovea. The correlation was determined between the extent of each perfusion grade and CFT. Results We found a significant negative correlation between the CFT and the FPA (r = 0.31, P = 0.006) and a significant positive correlation between the CFT and the PPA (r = 0.45, P < 0.001) in the three areas. The most significant correlations were found in the 2-DD area. Interestingly, the NPA has not correlated with the foveal thickness in any areas. Conclusion The areas of partial but not complete capillary loss seem to be responsible for the macular edema associated with BRVO. Treatments targeting leakage from the dilated capillaries in the PPA should be investigated.

Characteristics of the retinal surface after internal limiting membrane peeling in highly myopic eyes.

PURPOSE To document the characteristics of the retinal surface using en face optical coherence tomography (OCT) after internal limiting membrane (ILM) peeling during pars plana vitrectomy (PPV) for myopic macular diseases. DESIGN Retrospective, interventional case series. METHODS We examined 20 eyes of 20 patients with high myopia who underwent PPV with ILM peeling. The patients were followed for at least 6 months postoperatively, and OCT images were examined using spectral-domain OCT. To assess the incidence and patterns of the retinal dimple sign, we reconstructed en face OCT images and quantified the number of dimples during the follow-up period. RESULTS We identified 3 retinal dimple signs (ie, foveal-centered pattern, linear pattern, and paravascular pattern). The total numbers of retinal dimples at baseline and 1, 3, and 6 months postoperatively were 0, 31.8 ± 23.7, 47.95 ± 29.5, and 54.6 ± 30.7, respectively. There were significantly more dimples temporal to the macula compared to the other 3 quadrants at 1, 3, and 6 months postoperatively (P = .025, P = .0021, and P = .0060, respectively). However, the retinal sensitivities assessed by microperimetry were not correlated with the number of dimples. CONCLUSION Development of the retinal dimple sign after PPV and ILM peeling in highly myopic eyes was seen in all cases on en face OCT images. Although retinal dimples are benign, care should be taken after ILM peeling in highly myopic eyes.

Ligand-independent Tie2 dimers mediate kinase activity stimulated by high dose angiopoietin-1.

Background: Tie2 is essential for angiogenesis and vascular stabilization. Results: Tie2, but not Tie1, forms ligand-independent dimers on the cell surface. Conclusion: The inactive monomer mutant Tie2YIA/LAS decreases Ang1/Tie2 signaling. Significance: The Tie2 ligand-independent dimer induces strong phosphorylation upon high dose Ang1 binding. Tie2 is a receptor tyrosine kinase expressed on vascular endothelial cells (ECs). It has dual roles in promoting angiogenesis and stabilizing blood vessels, and it has been suggested that Tie2 forms dimers and/or oligomers in the absence of angiopoietin-1 (Ang1); however, the mechanism of ligand-independent dimerization of Tie2 and its biological significance have not been clarified. Using a bimolecular fluorescence complementation assay and a kinase-inactive Tie2 mutant, we show here that ligand-independent Tie2 dimerization is induced without Tie2 phosphorylation. Moreover, based on the fact that Tie1 never forms heterodimers with Tie2 in the absence of Ang1 despite having high amino acid sequence homology with Tie2, we searched for ligand-independent dimerization domains of Tie2 by reference to the difference with Tie1. We found that the YIA sequence of the intracellular domain of Tie2 corresponding to the LAS sequence in Tie1 is essential for this dimerization. When the YIA sequence was replaced by LAS in Tie2 (Tie2YIA/LAS), ligand-independent dimer was not formed in the absence of Ang1. When activation of Tie2YIA/LAS was induced by a high dose of Ang1, phosphorylation of Tie2 was limited compared with wild-type Tie2, resulting in retardation of activation of Erk downstream of Tie2. Therefore, these data suggest that ligand-independent dimerization of Tie2 is essential for a strong response upon stimulation with high dose Ang1.