Accurate prediction of disease-risk factors from volumetric medical scans by a deep vision model pre-trained with 2D scans.

The application of machine learning to tasks involving volumetric biomedical imaging is constrained by the limited availability of annotated datasets of three-dimensional (3D) scans for model training. Here we report a deep-learning model pre-trained on 2D scans (for which annotated data are relatively abundant) that accurately predicts disease-risk factors from 3D medical-scan modalities. The model, which we named SLIViT (for 'slice integration by vision transformer'), preprocesses a given volumetric scan into 2D images, extracts their feature map and integrates it into a single prediction. We evaluated the model in eight different learning tasks, including classification and regression for six datasets involving four volumetric imaging modalities (computed tomography, magnetic resonance imaging, optical coherence tomography and ultrasound). SLIViT consistently outperformed domain-specific state-of-the-art models and was typically as accurate as clinical specialists who had spent considerable time manually annotating the analysed scans. Automating diagnosis tasks involving volumetric scans may save valuable clinician hours, reduce data acquisition costs and duration, and help expedite medical research and clinical applications.

SLIViT: a general AI framework for clinical-feature diagnosis from limited 3D biomedical-imaging data.

We present SLIViT, a deep-learning framework that accurately measures disease-related risk factors in volumetric biomedical imaging, such as magnetic resonance imaging (MRI) scans, optical coherence tomography (OCT) scans, and ultrasound videos. To evaluate SLIViT, we applied it to five different datasets of these three different data modalities tackling seven learning tasks (including both classification and regression) and found that it consistently and significantly outperforms domain-specific state-of-the-art models, typically improving performance (ROC AUC or correlation) by 0.1-0.4. Notably, compared to existing approaches, SLIViT can be applied even when only a small number of annotated training samples is available, which is often a constraint in medical applications. When trained on less than 700 annotated volumes, SLIViT obtained accuracy comparable to trained clinical specialists while reducing annotation time by a factor of 5,000 demonstrating its utility to automate and expedite ongoing research and other practical clinical scenarios.

Role of Adrenomedullin 2/Intermedin in the Pathogenesis of Neovascular Age-Related Macular Degeneration.

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization-associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-ß2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration-related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

OCT Risk Factors for Development of Atrophy in Eyes with Intermediate Age-Related Macular Degeneration.

PURPOSE: To determine the frequency of multiple OCT biomarkers of intermediate age-related macular degeneration (iAMD) and their relationship with the development of complete retinal pigment epithelium and outer retinal atrophy (cRORA) after 2 years. DESIGN: Retrospective cohort study. PARTICIPANTS: This retrospective analysis included 330 eyes of 330 consecutive patients with iAMD in ≥ 1 eye who had 24 months of follow-up data. METHODS: Spectralis OCT volume scans (49 B-scans over 6 × 6 mm, automatic real time = 6, fovea-centered) at baseline were evaluated for the previously described iAMD biomarkers, including a high-central drusen volume (DV; ≥ 0.03 mm3), intraretinal hyper-reflective foci (IHRF), subretinal drusenoid deposits (SDDs), hypo-reflective drusen cores (hDCs), and a thin or thick (multilayered) double-layer sign (DLS). The age-related macular degeneration (AMD) status in the fellow eye was also assessed and classified as normal or early AMD, iAMD, exudative macular neovascularization, or cRORA. MAIN OUTCOME MEASURES: Incidence of cRORA, odds ratio for demographics, and OCT features. RESULTS: At month 24, 16.36% (54/330) of the iAMD eyes developed cRORA. Several baseline features, including high-central DV, IHRF, SDD, hDC, thin DLS, and cRORA in the fellow eye, were associated with a significantly greater risk for development of cRORA at 2 years. The odds ratio, 95% confidence interval, P value, and baseline frequencies of these biomarkers were DV (6.510, 2.467-17.176, P < 0.001, 49.1%), IHRF (12.763, 4.763-34.202, P < 0.001, 38.8%), SDD (2.307, 1.003-5.304, P = 0.049, 34.2%), hDC (3.012, 1.152-7.873, P = 0.024, 13.0%), thin DLS (4.517, 1.555-13.126, P = 0.006, 11.8%), and cRORA in the fellow eye (7.184, 1.938-26.623, P = 0.003, 8.2%). CONCLUSIONS: In addition to the 4 previously reported factors that are present in a significant proportion of iAMD (DV, IHRF, hDC, and SDD), a thin DLS and cRORA in the fellow eye were associated with an increased risk of progression to cRORA over 2 years. These biomarkers may aid in prognostication, risk stratification, and selection of patients for clinical trials. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Optical Coherence Tomography Biomarkers for Conversion to Exudative Neovascular Age-related Macular Degeneration.

PURPOSE: To identify optical coherence tomography (OCT) biomarkers, including thin and thick double-layer sign (DLS) for the progression from intermediate AMD (iAMD) to exudative macular neovascularization (MNV) over 24 months. DESIGN: Retrospective cohort study. METHODS: Setting: Retina consultants of Texas. PATIENT POPULATION: 458 eyes of 458 subjects with iAMD in at least 1 eye with 24 months of follow-up data. MAIN OUTCOMES MEASURES: The following biomarkers were assessed at baseline: high central drusen volume (≥0.03 mm3), intraretinal hyper-reflective foci (IHRF), subretinal drusenoid deposits, hyporeflective drusen cores, thick DLS, thin DLS, and central choroidal thickness. A binary logistic regression was computed to investigate the association between baseline OCT covariates and the conversion to exudative MNV within 24 months. In addition, fellow eye status was also included in the model. RESULTS: During follow-up, 18.1% (83 of 458) of eyes with iAMD progressed to exudative MNV. Thick DLS, IHRF, and fellow eye exudative MNV were found to be independent predictors for the development of exudative MNV within 2 years. The baseline frequencies, odds ratios, 95% confidence intervals, and P values for these biomarkers were as follows: thick DLS (9.6%, 4.339, 2.178-8.644; P < .001), IHRF (36.0%, 2.340, 1.396-3.922; P = 0.001), and fellow eye exudative MNV (35.8%, 1.694, 1.012-2.837; P = .045). CONCLUSIONS: Thick DLS, IHRF, and fellow eye exudative MNV were associated with an increased risk of progression from iAMD to exudative MNV. These biomarkers, which are readily identified by the review of OCT volume scans, may aid in risk prognostication for patients and for identifying patients for early intervention trials.

Morphological Changes in the Foveal Avascular Zone after Panretinal Photocoagulation for Diabetic Retinopathy Using OCTA: A Study Focusing on Macular Ischemia.

Background and objectives: This study aimed to analyze the morphological changes in the foveal avascular zone (FAZ) after panretinal photocoagulation (PRP) in patients with diabetic retinopathy, with a particular focus on the presence or absence of comorbid diabetic macular ischemia (DMI), using optical coherence tomography angiography (OCTA). Materials and Methods: Treatment-naïve 25 eyes of 16 patients who received PRP were examined in this retrospective case series. FAZ area, perimeter, and circularity were calculated on a 3 × 3-mm en-face OCTA image before PRP (baseline) and 1 and 3 months after PRP. The patients were divided into two groups according to coexisting DMI, and each group was statistically analyzed. Results: In patients with DMI (9 eyes), FAZ area significantly decreased from the baseline to 3 months after PRP (0.86 ± 0.56 to 0.61 ± 0.31 mm2, p = 0.018), whereas FAZ perimeter and circularity remained unchanged following treatment (p = 0.569 and 0.971, respectively). In patients without DMI (16 eyes), FAZ parameters did not show statistically significant changes across the 3-month follow-up period. Conclusion: PRP significantly reduces FAZ area in patients with DMI.

A Case of Diagnosis of Occipital Lobe Epilepsy Complicated by Right Hemianopsia Associated with Left Occipital Lobe Cerebral Infarction.

We report a case of occipital lobe epilepsy (OLE) in a patient with occipital lobe stroke whose diagnosis was complicated by homonymous hemianopsia. An 81-year-old woman presented with a complaint of "blurred vision" on the right side and was kept under outpatient observation at the Hirabayashi Eye Clinic for homonymous lower right hemianopsia, glaucoma, and post-cataract surgery. Her past medical history included hypertension, angina pectoris, atrial fibrillation, diabetes mellitus, and left occipital lobe cerebral infarction. The corrected visual acuity and intraocular pressure were 20/16 and 12 mm Hg and 20/20 and 13 mm Hg in the right and left eye, respectively, and no change was observed in the fundus and visual field defect; hence, the patient was placed under observation. Two days later, the patient voluntarily visited a neurosurgical hospital and underwent magnetic resonance imaging. No abnormalities were detected other than the left obsolete occipital lobe stroke. Five days later, she returned to our clinic because she felt "something wobbly" on her right side. Upon examination, we suspected a transient ischemic attack based on the wobbling, closed eyelids, and loss of consciousness, and referred her to the same neurosurgical hospital. Electroencephalography (EEG) revealed spikes and waves with occipital lobe predominance, and the diagnosis of OLE was made. The patient had right-sided homonymous hemianopsia owing to left occipital lobe cerebral infarction and "blurred vision" on the same side. Thus, it is inferred that EEG is imperative for ruling out epileptic seizures.

Adrenomedullin-Receptor Activity-Modifying Protein 2 System Ameliorates Subretinal Fibrosis by Suppressing Epithelial-Mesenchymal Transition in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti-vascular endothelial growth factor drugs used to treat AMD carry the risk of inducing subretinal fibrosis. We investigated the use of adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. The therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with those in wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-ß and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, transforming growth factor-ß and C-X-C chemokine receptor type 4 (CXCR4) inhibitors eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.

Prediction of post-treatment retinal sensitivity by baseline retinal perfusion density measurements in eyes with branch retinal vein occlusion.

In this study, we investigated the longitudinal correlation between macular sensitivity and perfusion density (PD) in retinas affected by branch retinal vein occlusion. Retinal sensitivity was measured using microperimetry and PD was measured by optical coherence tomography angiography. We also investigated the possibility that the PD, 1 month after anti-vascular endothelial growth factor (VEGF) treatment, is a predictor of retinal sensitivity after 1 year of successful macular oedema management with anti-VEGF. The correlation between measurements of retinal sensitivity and PD at baseline (1 M) and at 6 and 12 months were investigated. There was a significant positive correlation between retinal sensitivity and PD at all time points (baseline (1 M), r = 0.67, P < 0.0001; 6 months, r = 0.59, P < 0.0001; 12 months, r = 0.62, P < 0.0001) and between the PD at 1 month and retinal sensitivity at 12 months (r = 0.63, P < 0.0001). Unlike in areas that showed a mild to moderate decline in PD, retinal sensitivity in areas where the decrease in PD was severe at baseline did not show significant improvement with treatment over time. These findings suggest that the PD value measured using optical coherence tomography angiography at or soon after the baseline can predict retinal sensitivity after 1 year of anti-VEGF treatment.

Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.

Vitreoretinal Interface Slab in OCT Angiography for Detecting Diabetic Retinal Neovascularization.

PURPOSE: To compare neovascularization identified in proliferative diabetic retinopathy (PDR) eyes by widefield swept-source (SS) OCT angiography (OCTA) using vitreoretinal interface (VRI) slab images, composed by automated and manual segmentation, with that identified by fluorescein angiography (FA). DESIGN: Retrospective study. PARTICIPANTS: Forty-two eyes of 30 treatment-naïve PDR patients who visited the outpatient clinic of the Department of Ophthalmology, Shinshu University, from June 2018 through October 2019. METHODS: All patients underwent comprehensive ophthalmologic examinations, including SS-OCTA and FA. MAIN OUTCOME MEASURES: Neovascularization detected by en face SS-OCTA 15 × 15-mm VRI slab images and by FA in the same 15 × 15-mm areas were compared in terms of number and structure. RESULTS: Among 100 neovascularizations detected by FA, 73 also were visualized as neovascularization in SS-OCTA VRI slab images using automated segmentation. The sensitivity of VRI slab images for detecting neovascularization was 73%. Among the remaining 27 neovascularizations detected only by FA, but not by VRI slab, 15 were intraretinal microvascular abnormalities with fluorescence leakage, 1 was a diabetic papillopathy, and 11 were flat neovascularizations on the internal limiting membrane surface that were missed because of segmentation error. Conversely, among the 98 neovascularizations detected on VRI slab images, 25 were not detected as neovascularizations by FA. They included 9 small neovascularizations that exhibited too little leakage on FA and 16 false-positive results that were the result of segmentation errors. After reconstruction of SS-OCTA VRI slab images by means of manual segmentation, the sensitivity of VRI slab images for detecting neovascularizations increased to 84%. CONCLUSIONS: The efficacy of SS-OCTA VRI slab images for detecting neovascularizations in PDR was comparable with that of FA. Swept-source OCTA VRI slab images may be better than FA for identifying intraretinal microvascular abnormalities and diabetic papillopathy from neovascularizations. Notably, however, FA and SS-OCTA VRI slab images demonstrated differences in identification efficacy in cases of small and flat neovascularizations. Further exploration of SS-OCTA technology is warranted to address this issue.

Development of a Novel Model of Central Retinal Vascular Occlusion and the Therapeutic Potential of the Adrenomedullin-Receptor Activity-Modifying Protein 2 System.

Central retinal vein occlusion (CRVO) is an intractable disease that causes visual acuity loss with retinal ischemia, hemorrhage, and edema. In this study, we developed an experimental CRVO model in mice and evaluated the therapeutic potential of the pleiotropic peptide adrenomedullin (ADM) and its receptor activity-modifying protein 2 (RAMP2). The CRVO model, which had phenotypes resembling those seen in the clinic, was produced by combining i.p. injection of Rose bengal, a photoactivator dye enhancing thrombus formation, with laser photocoagulation. Retinal vascular area, analyzed using fluorescein angiography and fluorescein isothiocyanate-perfused retinal flat mounts, was decreased after induction of CRVO but gradually recovered from day 1 to 7. Measurements of retinal thickness using optical coherence tomography and histology revealed prominent edema early after CRVO, followed by gradual atrophy. Reperfusion after CRVO was diminished in Adm and Ramp2 knockout (KO) mice but was increased by exogenous ADM administration. CRVO also increased expression of a coagulation factor, oxidative stress markers, and a leukocyte adhesion molecule in both wild-type and Adm KO mice, and the effect was more pronounced in Adm KO mice. Using retinal capillary endothelial cells, ADM was found to directly suppress retinal endothelial injury. The retinoprotective effects of the Adm-Ramp2 system make it a novel therapeutic target for the treatment of CRVO.

RAMP3 deficiency enhances postmenopausal obesity and metabolic disorders.

There is a marked increase in the incidence of visceral adiposity and insulin resistance among women following menopause. Adrenomedullin (AM) is an endogenous peptide first identified as a vasodilator, but now known to exert a variety of physiological effects. RAMP3 is a receptor activity-modifying protein that binds to the AM receptor (calcitonin receptor-like receptor). As expression of both AM and RAMP3 is reportedly activated by estrogen, we hypothesized that RAMP3 is crucially involved in the pathophysiology of postmenopausal obesity. To test this idea, we compared the effects of ovariectomy (OVX) and a high-fat diet for 10 weeks (a model of postmenopausal obesity) between RAMP3 knockout (RAMP3-/-) and wild-type mice. RAMP3-/- OVX mice exhibited greater obesity and adipose tissue weight gain as compared to wild-type OVX mice. RAMP3-/- OVX mice also exhibited higher serum insulin levels. In periuterine WAT from RAMP3-/- OVX mice, expression of lipolysis-related factors was lower and expression of inflammation-related factors was higher than in wild-type OVX mice. Hepatic steatosis was also exacerbated in RAMP3-/- OVX. Notably, expression of the membrane-type estrogen receptor GPR30 was downregulated in periuterine WAT from RAMP3-/- OVX mice. These findings raise the possibility that a GPR30-RAMP3 interaction is involved in the pathophysiology of postmenopausal obesity and suggest RAMP3 plays a key role in the regulation of energy metabolism and exerts a hepatoprotective effect in this model of postmenopausal obesity. RAMP3 may thus be a useful therapeutic target for treatment of postmenopausal obesity and metabolic disorders.

Endogenous calcitonin gene-related peptide suppresses ischemic brain injuries and progression of cognitive decline.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide and produced by alternative splicing of the transcript of the calcitonin/CGRP gene. Originally identified as a strong vasodilatory and hypotensive peptide, CGRP is now known to be a pleiotropic molecule distributed in various organs, including the brain. METHOD: In this study, we used CGRP knockout mice (CGRP-/-) to examine the actions of endogenous CGRP during cerebral ischemia. To induce acute and chronic cerebral ischemia, mice were subjected to middle cerebral artery occlusion (MCAO) and bilateral common carotid artery stenosis (BCAS). RESULTS: In the cerebral cortex of wild-type mice, CGRP expression was upregulated after acute infarction. In CGRP-/- subjected to MCAO or BCAS, recovery of cerebral blood flow was slower and exhibited more extensive neuronal cell death. Expression of the inflammatory cytokines was higher in CGRP-/- than wild type in the acute phase of ischemia. Pathological analysis during the chronic phase revealed more extensive neuronal cell loss and demyelination and higher levels of oxidative stress in CGRP-/- than wild-type. CGRP-/- also showed less compensatory capillary growth. In an eight-arm radial maze test, CGRP-/- exhibited poorer reference memory than wild-type. On the other hand, CGRP administration promoted cerebral blood flow recovery after cerebral ischemia. We also found that CGRP directly inhibited the cell death of primary cortical neurons. CONCLUSION: These results indicate endogenous CGRP is protective against ischemia-induced neuronal cell injury. CGRP could, thus, be a novel candidate for use in the treatment of both cerebral ischemia and progression of cognitive decline.

Endogenous Calcitonin Gene-Related Peptide Regulates Lipid Metabolism and Energy Homeostasis in Male Mice.

Calcitonin gene-related peptide (CGRP) is a bioactive peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. CGRP is largely distributed in the cardiovascular and nervous systems, where it acts as a regulatory factor. CGRP is also expressed in organs and tissues involved in metabolic regulation, including white adipose tissue (WAT), where its function is largely unknown. In this study, we examined the effects of endogenous CGRP on metabolic function. When we administered a high-fat diet to CGRP-specific knockout (CGRP-/-) and wild-type (WT) mice for 10 weeks, we observed that food intake did not differ between the two groups, but body weight and visceral fat weight were significantly lower in CGRP-/- mice. Fatty liver changes were less severe in CGRP-/- mice, which also showed lower serum insulin and leptin levels. Glucose tolerance and insulin sensitivity were better in CGRP-/- than WT mice, and expired gas analysis revealed greater oxygen consumption by CGRP-/- mice. Adipocyte hypertrophy was suppressed in CGRP-/- mice, while expression of ß-3-adrenergic receptor, hormone-sensitive lipase and adiponectin was enhanced. Isoproterenol-induced glycerol release from WAT was higher in CGRP-/- than WT mice, and CGRP-/- mice showed elevated sympathetic nervous activity. ß-receptor-blockade canceled the beneficial effects of CGRP deletion on obesity. These results suggest that, in addition to its actions in the cardiovascular system, endogenous CGRP is a key regulator of metabolism and energy homeostasis in vivo.

Vasoprotective Activities of the Adrenomedullin-RAMP2 System in Endothelial Cells.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after coronary intervention. We previously described the essential angiogenic function of the adrenomedullin (AM)-receptor activity-modifying protein (RAMP) 2 system. In the present study, we assessed the vasoprotective actions of the endogenous AM-RAMP2 system using a wire-induced vascular injury model. We found that neointima formation and vascular smooth muscle cell proliferation were enhanced in RAMP2+/- male mice. The injured vessels from RAMP2+/- mice showed greater macrophage infiltration, inflammatory cytokine expression, and oxidative stress than vessels from wild-type mice and less re-endothelialization. After endothelial cell-specific RAMP2 deletion in drug-inducible endothelial cell-specific RAMP2-/- (DI-E-RAMP2-/-) male mice, we observed markedly greater neointima formation than in control mice. In addition, neointima formation after vessel injury was enhanced in mice receiving bone marrow transplants from RAMP2+/- or DI-E-RAMP2-/- mice, indicating that bone marrow-derived cells contributed to the enhanced neointima formation. Finally, we found that the AM-RAMP2 system augmented proliferation and migration of endothelial progenitor cells. These results demonstrate that the AM-RAMP2 system exerts crucial vasoprotective effects after vascular injury and could be a therapeutic target for the treatment of vascular diseases.

Adrenomedullin Suppresses Vascular Endothelial Growth Factor-Induced Vascular Hyperpermeability and Inflammation in Retinopathy.

Diabetic macular edema (DME) is caused by blood-retinal barrier breakdown associated with retinal vascular hyperpermeability and inflammation, and it is the major cause of visual dysfunction in diabetic retinopathy. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilator. ADM is expressed in the eyes and is up-regulated in various eye diseases, although the pathophysiological significance is largely unknown. We investigated the effect of ADM on DME. In Kimba mice, which overexpress human vascular endothelial growth factor in their retinas, the capillary dropout, vascular leakage, and vascular fragility characteristic of diabetic retinopathy were observed. Intravitreal or systemic administration of ADM to Kimba mice ameliorated both the capillary dropout and vascular leakage. Evaluation of the transendothelial electrical resistance and fluorescein isothiocyanate-dextran permeability of an endothelial cell monolayer using TR-iBRB retinal capillary endothelial cells revealed that vascular endothelial growth factor enhanced vascular permeability but that co-administration of ADM suppressed the effect, in part by enhancing tight junction formation between endothelial cells. In addition, a comprehensive PCR array analysis showed that ADM administration suppressed various molecules related to inflammation and NF-κB signaling within retinas. From these results, we suggest that by exerting inhibitory effects on retinal inflammation, vascular permeability, and blood-retinal barrier breakdown, ADM could serve as a novel therapeutic agent for the treatment of DME.

Effect of leaking perifoveal microaneurysms on resolution of diabetic macular edema treated by combination therapy using anti-vascular endothelial growth factor and short pulse focal/grid laser photocoagulation.

PURPOSE: The effect of combination therapy using intravitreal ranibizumab (IVR) injections and short pulse focal/grid laser photocoagulation was evaluated for the treatment of diabetic macular edema (DME). METHODS: The current investigation was a preliminary single-arm, open-label, prospective clinical study conducted on 21 eyes at 4 sites in Japan. Treatment protocol consisted of two phases. The induction IVR phase included two monthly IVRs followed by PRN IVR phase in which additional IVR was administered if the central macular thickness (CMT) exceeded 300 µm. One week after each IVR in both phases, short pulse focal/grid laser was delivered to treat residual leakage outside of the fovea (>500 µm) and reduce edema fluid influx. At the 6-month endpoint, the effects of treatment were examined in terms of best corrected visual acuity (BCVA), CMT, and required number of IVR injections in eyes with or without perifoveal leaking microaneurysms (MAs). RESULTS: In eyes with initial BCVA ≤70 letters, mean BCVA was significantly ameliorated by 7.0 ± 7.4 letters (P = 0.0324) and mean CMT improved significantly by 174.8 ± 105.0 µm (P = 0.0005). Both BCVA improvement (P = 0.8693) and CMT reduction (P = 0.9336) were comparable between MA(-) and MA(+) groups. The MA(-) group required significantly fewer PRN-IVR injections than did the MA(+) group over the 6-month study period (mean 3.4 ± 1.6 vs. 5.3 ± 0.9, median 3.0 vs. 5.5; P = 0.0229). CONCLUSIONS: Short pulse focal/grid laser photocoagulation could reduce the number of IVR injections required to resolve macular edema and increase BCVA in a possible mechanism of reduced influx of edema fluid into the foveal area in eyes without apparent perifoveal microaneurysms.

The endothelial adrenomedullin-RAMP2 system regulates vascular integrity and suppresses tumour metastasis.

AIMS: Controlling vascular integrity is expected to be a novel therapeutic target of cancers as well as cardiovascular diseases. Adrenomedullin (AM) and its receptor-modulating protein, RAMP2, have been identified as essential mediators of cardiovascular homeostasis. In this study, we used inducible vascular endothelial cell-specific RAMP2 knockout (DI-E-RAMP2(-/-)) mice to clarify the contribution made by the endogenous AM-RAMP2 system to angiogenesis and metastasis. METHODS AND RESULTS: Subcutaneously transplanted sarcoma or melanoma cells showed less growth and angiogenesis in DI-E-RAMP2(-/-) than in control mice. On the other hand, after the transplantation of B16BL6 melanoma cells into hindlimb footpads, spontaneous metastasis to the lung was enhanced in DI-E-RAMP2(-/-) mice. Early after RAMP2 gene deletion, DI-E-RAMP2(-/-) mice showed enhanced vascular permeability, endothelial-mesenchymal transition (EndMT)-like change, and systemic oedema. Within the lungs of DI-E-RAMP2(-/-) mice, pulmonary endothelial cells were deformed, and inflammatory cells infiltrated the vessel walls and expressed the chemotactic factors S100A8/9 and SAA3, which attract tumour cells and mediate the formation of a pre-metastatic niche. Conversely, the overexpression of RAMP2 suppressed tumour cell adhesion to endothelial cells, tumour metastasis, and improved survival. CONCLUSION: These findings indicate that the AM-RAMP2 system regulates vascular integrity, whereas RAMP2 deletion promotes vascular permeability and EndMT-like change within primary lesions and formation of pre-metastatic niches in distant organs by destabilizing the vascular structure and inducing inflammation. Vascular integrity regulated by the AM-RAMP2 system could thus be a hopeful therapeutic target for suppressing tumour metastasis.