Muneo Yamaguchi

ROCK-Isoform-Specific Polarization of Macrophages Associated with Age-Related Macular Degeneration

Age is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

Increased vitreous concentrations of MCP-1 and IL-6 after vitrectomy in patients with proliferative diabetic retinopathy: possible association with postoperative macular oedema

Purpose To determine whether vitreal concentrations of MCP-1, IL-6 and IL-8 are altered after vitrectomy in patients with proliferative diabetic retinopathy (PDR) and to investigate whether the altered levels of these cytokines are associated with postoperative macular oedema. Methods Vitreous samples were collected from 36 eyes of 33 patients with PDR before pars plana vitrectomy without intraocular lens (IOL) implantation, and also from the same 36 eyes during IOL implantation surgery approximately 7 months after the initial vitrectomy. Levels of MCP-1, IL-6, IL-8 and vascular endothelial growth factor were measured by flow cytometry using cytometric bead array (CBA) technology. Results The mean vitreous levels of MCP-1, IL-6 and IL-8 in the samples collected before vitrectomy were significantly higher in patients with PDR than in control patients (p<0.0001). The levels of MCP-1 and IL-6 in the samples collected at the time of IOL implantation were significantly higher than those collected before vitrectomy (p<0.05). In contrast, the level of IL-8 was significantly lower after vitrectomy (p<0.05). The levels of IL-6 and IL-8, but not MCP-1, in the vitreous from eyes with PDR were inversely correlated with the interval between the initial vitrectomy and the time of implantation surgery. Among the vitrectomised patients, the mean vitreous level of MCP-1 in eyes with diabetic macular oedema (DME) was significantly higher than in those without DME (p=0.028). Conclusions The elevated levels of MCP-1 and IL-6 may indicate prolonged inflammation even after successful vitrectomy, which can cause postoperative DME.

Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia.

PURPOSE In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia. METHODS In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. RESULTS Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 μmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 μmol/L and 6.8 to 14.8 μmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion. CONCLUSIONS Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.

M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy

PURPOSE To investigate the roles played by M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR). METHODS Oxygen-induced retinopathy was induced in C57BL/6J mice by exposing postnatal day seven (P7) pups to 75% oxygen and then returning them to room air at P12. Real-time RT-PCR and immunofluorescence staining were used to assess the levels and distributions of different macrophage markers. Bone marrow-derived M1 and M2 macrophages and mannosylated clodronate liposomes (MCLs) were injected into the vitreous on P12 to examine the effects at P17. M2 macrophages were cocultured with human retinal endothelial cells (HRECs) to examine their effects on proliferation and tube formation. RESULTS The results showed that the M2 macrophages, rather than M1 phenotype, were highly expressed in OIR mice. The number of M2 macrophages had increased significantly at P17, and the increase was closely associated with the presence of neovascular tufts in the OIR retinas. Selective depletion of M2 macrophages suppressed the pathological neovascularization and promoted physiological revascularization. In contrast, intravitreal injection of bone marrow-derived M2 macrophages or the culture supernatants promoted pathological neovascularization and inhibited physiological revascularization. In an in vitro coculture system, M2-polarized macrophages significantly promoted proliferation and tube formation of HRECs. CONCLUSIONS These results indicated that M2 macrophages, rather than M1, play an important role in promoting retinal pathological neovascularization probably by producing secreted factors. Thus, targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization.

Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy

Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization.

High-Resolution Imaging by Adaptive Optics Scanning Laser Ophthalmoscopy Reveals Two Morphologically Distinct Types of Retinal Hard Exudates

Histological studies from autopsy specimens have characterized hard exudates as a composition of lipid-laden macrophages or noncellular materials including lipid and proteinaceous substances (hyaline substances). However, the characteristics of hard exudates in living patients have not been examined due to insufficient resolution of existing equipment. In this study, we used adaptive optics scanning laser ophthalmoscopy (AO-SLO) to examine the characteristics of hard exudates in patients with retinal vascular diseases. High resolution imaging using AO-SLO enables morphological classification of retinal hard exudates into two types, which could not be distinguished either on fundus examination or by spectral domain optical coherence tomography (SD-OCT). One, termed a round type, consisted of an accumulation of spherical particles (average diameter of particles: 26.9 ± 4.4 μm). The other, termed an irregular type, comprised an irregularly shaped hyper-reflective deposition. The retinal thickness in regions with round hard exudates was significantly greater than the thickness in regions with irregular hard exudates (P = 0.01 →0.02). This differentiation of retinal hard exudates in patients by AO-SLO may help in understanding the pathogenesis and clinical prognosis of retinal vascular diseases.

Optical coherence tomography angiography reveals spatial bias of macular capillary dropout in diabetic retinopathy

Purpose Our purpose is to evaluate the spatial bias of macular capillary dropout accompanying diabetic retinopathy (DR) using optical coherence tomography angiography (OCTA). Methods This study included 47 patients with diabetes and 29 healthy individuals who underwent OCTA. Retinal capillary flow density (FD) of 2.6 × 2.6 or 5.2 × 5.2 mm foveal area as well as the four divided areas (superior, inferior, temporal, nasal) without a foveal avascular zone (FAZ) at the superficial capillary plexus and deep capillary plexus (DCP) were measured respectively using ImageJ and NI Vision. Spatial biases of FD (orientation bias ratio and hierarchical bias ratio) and the correlation between FAZ and FD were examined. Results OCTA showed focal capillary dropout in DR patients. The orientation bias of FD was significantly higher in NPDR compared to NDR in the DCP (P = 0.03). The hierarchical bias of FD was significantly shifted to a DCP dominance with progression of DR (P < 0.01). In addition, the FD and FAZ area were significantly inversely correlated in both plexus in DR patients but not in healthy subjects (P < 0.01). Conclusions Area-divided OCTA quantification shows the appearance of spatial biases of macular capillary dropout with the onset of DR, suggesting that DR-related macular capillary dropout occurs locally and randomly. Future studies are necessary to determine the clinical relevance of the spatial pattern of capillary dropout in DR.

Different distributions of M1 and M2 macrophages in a mouse model of laser-induced choroidal neovascularization

Choroidal neovascularization (CNV) is a serious complication of age-related macular degeneration. The aim of the present study was to investigate the expression and distribution of M1 and M2 macrophages in a laser-induced CNV adult mouse model. The mRNA expression levels of M1, M2 and pan macrophage markers, and macrophage-associated angiogenic cytokines, were determined by reverse transcription-quantitative polymerase chain reaction. Immunofluorescence studies were performed to determine the location of the macrophages. The expression levels of M1 macrophage markers increased to a greater extent compared with M2 markers in the retinal pigment epithelium (RPE)-choroid complexes following laser photocoagulation. By contrast, the expression levels of M2 macrophage markers increased primarily in the retinas. Immunofluorescence studies revealed that the increased number of cluster of differentiation (CD)206-positive cells were located primarily in the retina, whereas the CD80-positive cells were located around the site of CNVs in the RPE-choroid. In addition, the M1-associated cytokines increased to a greater extent in the RPE-choroid complexes, whereas the M2-associated cytokines were highly expressed in the retinas. These findings indicate that M1 and M2 macrophage numbers increased following CNV; however, the locations were different in this mouse model of laser-induced CNV. The results of the present study suggest that M1 macrophages have a more direct role in inhibiting the development of CNV.

Rho-Kinase/ROCK as a Potential Drug Target for Vitreoretinal Diseases

Rho-associated kinase (Rho-kinase/ROCK) was originally identified as an effector protein of the G protein Rho. Its involvement in various diseases, particularly cancer and cardiovascular disease, has been elucidated, and ROCK inhibitors have already been applied clinically for cerebral vasospasm and glaucoma. Vitreoretinal diseases including diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinoapthy are still a major cause of blindness. While anti-VEGF therapy has recently been widely used for vitreoretinal disorders due to its efficacy, attention has been drawn to new unmet needs. The importance of ROCK in pathological vitreoretinal conditions has also been elucidated and is attracting attention as a potential therapeutic target. ROCK is involved in angiogenesis and hyperpermeability and also in the pathogenesis of various pathologies such as inflammation and fibrosis. It has been expected that ROCK inhibitors will become new molecular target drugs for vitreoretinal diseases. This review summarizes the recent progress on the mechanisms of action of ROCK and their applications in disease treatment.

Comparison of the Effectiveness of Intravitreal Ranibizumab for Diabetic Macular Edema in Vitrectomized and Nonvitrectomized Eyes

Purpose: To compare the effectiveness of intravitreal ranibizumab (IVR) for diabetic macular edema (DME) between eyes with and without previous vitrectomy. Procedures: We prospectively assessed the best-corrected visual acuity (BCVA) and central macular thickness (CMT) after IVR for 6 months. Results: There were no significant differences in the baseline BCVA and CMT between both groups. In the nonvitrectomized group (n = 15), the mean changes of BCVA and CMT from baseline to month 6 were significant (p < 0.01). In the vitrectomized group (n = 10), the improvement appeared to be slower, and the mean BCVA improvement was not significant (p = 0.5), although the mean CMT decrease was significant (p < 0.05). There were no significant differences in the mean changes of BCVA and CMT between both groups at 6 months. Conclusions: The difference in the effectiveness of IVR between both groups was not significant. IVR can be a treatment option even for vitrectomized DME eyes.