Yasuhiro Iesato

Vascular Endothelial Adrenomedullin-RAMP2 System Is Essential for Vascular Integrity and Organ Homeostasis

Background— Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. Methods and Results— We generated endothelial cell–specific RAMP2 and AM knockout mice (E-RAMP2−/− and E-AM−/−). Most E-RAMP2−/− mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2−/− mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2−/− mice (DI-E-RAMP2−/−) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. Conclusions— Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage.

Correlation between diabetic retinopathy severity and elevated skin autofluorescence as a marker of advanced glycation end-product accumulation in type 2 diabetic patients.

AIMS We evaluated skin autofluorescence (AF) as a marker of tissue advanced glycation end-product (AGE) accumulation and examined whether it was related to the prevalence and severity of diabetic retinopathy (DR) and of diabetic macular edema (DME) in patients with type 2 diabetes mellitus (DM). METHODS This study included 138 type 2 DM patients consisting of 31 patients with proliferative DR, 71 patients with non-proliferative DR, and 36 patients without retinopathy, in addition to 111 non-DM control subjects. At the time of skin AF and HbA1c measurement, self-assessed duration of DM was also determined. DR and DME stages were classified according to international guidelines. RESULTS Skin AF was significantly increased in patients with DM as compared with non-DM controls. Furthermore, skin AF was correlated with the severity of DR, whereas single measurement of HbA1c and self-assessed DM duration were not. None of these 3 factors showed a correlation with DME prevalence or severity. CONCLUSIONS Skin AF levels, which can be measured non-invasively on a screening basis without skin biopsy or blood sampling, have a greater predictive ability for the presence and severity of DR than single measurement of HbA1c or self-assessed DM duration in patients with type 2 DM.

Novel Regulation of Cardiac Metabolism and Homeostasis by the Adrenomedullin-Receptor Activity-Modifying Protein 2 System

Adrenomedullin (AM) was identified as a vasodilating and hypotensive peptide mainly produced by the cardiovascular system. The AM receptor calcitonin receptor-like receptor associates with receptor activity-modifying protein (RAMP), one of the subtypes of regulatory proteins. Among knockout mice (−/−) of RAMPs, only RAMP2−/− is embryonically lethal with cardiovascular abnormalities that are the same as AM−/−. This suggests that the AM-RAMP2 system is particularly important for the cardiovascular system. Although AM and RAMP2 are highly expressed in the heart from embryo to adulthood, their analysis has been limited by the embryonic lethality of AM−/− and RAMP2−/−. For this study, we generated inducible cardiac myocyte-specific RAMP2−/− (C-RAMP2−/−). C-RAMP2−/− exhibited dilated cardiomyopathy-like heart failure with cardiac dilatation and myofibril disruption. C-RAMP2−/− hearts also showed changes in mitochondrial structure and downregulation of mitochondria-related genes involved in oxidative phosphorylation, &bgr;-oxidation, and reactive oxygen species regulation. Furthermore, the heart failure was preceded by changes in peroxisome proliferator-activated receptor-&ggr; coactivator 1&agr; (PGC-1&agr;), a master regulator of mitochondrial biogenesis. Metabolome and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) imaging analyses revealed early downregulation of cardiolipin, a mitochondrial membrane-specific lipid. Furthermore, primary-cultured cardiac myocytes from C-RAMP2−/− showed reduced mitochondrial membrane potential and enhanced reactive oxygen species production in a RAMP2 deletion–dependent manner. C-RAMP2−/− showed downregulated activation of cAMP response element binding protein (CREB), one of the main regulators of mitochondria-related genes. These data demonstrate that the AM-RAMP2 system is essential for cardiac metabolism and homeostasis. The AM-RAMP2 system is a promising therapeutic target of heart failure.

En face swept-source optical coherence tomography detecting thinning of inner retinal layers as an indicator of capillary nonperfusion.

Purpose To report en face swept-source optical coherence tomography (SS-OCT) as a noninvasive detection modality for regions of retinal layer thinning that are potential indicators of retinal nonperfusion areas (NPAs) in patients with branch retinal vein occlusion (BRVO). Methods Thirty-one eyes of 46 patients with BRVO showing a definite retinal NPA larger than 1 disc in diameter within the vascular arcades were included in this study. We calculated the overlapping correspondence ratio between presumed NPA, which appeared as a dark area in en face SS-OCT, and definite NPA in corresponding fluorescein angiography (FA) images. The correlation between brightness in en face SS-OCT images and corresponding ganglion cell complex (GCC) thickness determined by SD-OCT GCC maps was evaluated as well. Results Measured NPA size in FA and presumed NPA size in en face SS-OCT showed strong correlation (r = 0.746, p<0.001) and colocalization. Reliable agreement between the 2 methods was confirmed by size comparisons (p = 0.11), with an overlapping correspondence ratio of 0.73. Conclusions The results suggest that en face SS-OCT is a noninvasive and relatively reliable method for delineating retinal thinning as an indicator of NPAs in eyes with BRVO. Because SS-OCT can be performed without mydriasis, this procedure may be an option for evaluating NPAs on a screening basis and during follow-up to reduce the number of FA examinations that carry very rare, but potentially fatal, allergic side effects.

Endogenous CGRP protects against neointimal hyperplasia following wire-induced vascular injury.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after percutaneous coronary intervention. Calcitonin gene-related peptide (CGRP) is produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. Originally identified as a strongly vasodilatory neuropeptide, CGRP is now known to be a pleiotropic peptide widely distributed in various organs and tissues. Our aim was to investigate the possibility that CGRP acts as an endogenous vasoprotective molecule. We compared the effect of CGRP deficiency on neointimal formation after wire-induced vascular injury in wild-type and CGRP knockout (CGRP-/-) mice. We found that neointimal formation after vascular injury was markedly enhanced in CGRP-/- mice, which also showed a higher degree of oxidative stress, as indicated by reduced expression of nitric oxide synthase, increased expression of p47phox, and elevated levels of 4HNE, as well as greater infiltration of macrophages. In addition, CGRP-deficiency led to increased vascular smooth muscle cell (VSMC) proliferation within the neointima. By contrast, bone marrow-derived cells had little or no effect on neointimal formation in CGRP-/-mice. In vitro analysis showed that CGRP-treatment suppressed VSMC proliferation, migration, and ERK1/2 activity. These results clearly demonstrate that endogenous CGRP suppresses the oxidative stress and VSMC proliferation induced by vascular injury. As a vasoprotective molecule, CGRP could be an important therapeutic target in cardiovascular disease.

Adrenomedullin-RAMP2 system is crucially involved in retinal angiogenesis.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Adrenomedullin-RAMP2 System Suppresses ER Stress-Induced Tubule Cell Death and Is Involved in Kidney Protection

Various bioactive peptides have been implicated in the homeostasis of organs and tissues. Adrenomedullin (AM) is a peptide with various bioactivities. AM-receptor, calcitonin-receptor-like receptor (CLR) associates with one of the subtypes of the accessory proteins, RAMPs. Among the RAMP subisoforms, only RAMP2 knockout mice (−/−) reproduce the phenotype of embryonic lethality of AM−/−, illustrating the importance of the AM-RAMP2-signaling system. Although AM and RAMP2 are abundantly expressed in kidney, their function there remains largely unknown. We used genetically modified mice to assess the pathophysiological functions of the AM-RAMP2 system. RAMP2+/− mice and their wild-type littermates were used in a streptozotocin (STZ)-induced renal injury model. The effect of STZ on glomeruli did not differ between the 2 types of mice. On the other hand, damage to the proximal urinary tubules was greater in RAMP2+/−. Tubular injury in RAMP2+/− was resistant to correction of blood glucose by insulin administration. We examined the effect of STZ on human renal proximal tubule epithelial cells (RPTECs), which express glucose transporter 2 (GLUT2), the glucose transporter that specifically takes up STZ. STZ activated the endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK). AM suppressed PERK activation, its downstream signaling, and CCAAT/enhancer-binding homologous protein (CHOP)-induced cell death. We confirmed that the tubular damage was caused by ER stress-induced cell death using tunicamycin (TUN), which directly evokes ER stress. In RAMP2+/− kidneys, TUN caused severe injury with enhanced ER stress. In wild-type mice, TUN-induced tubular damage was reversed by AM administration. On the other hand, in RAMP2+/−, the rescue effect of exogenous AM was lost. These results indicate that the AM-RAMP2 system suppresses ER stress-induced tubule cell death, thereby exerting a protective effect on kidney. The AM-RAMP2 system thus has the potential to serve as a therapeutic target in kidney disease.

Detection of Fovea-Threatening Diabetic Macular Edema by Optical Coherence Tomography to Maintain Good Vision by Prophylactic Treatment

Background/Aims: To establish a screening and treatment method for fovea-threatening diabetic macular edema (DME) using spectral-domain optical coherence tomography (SD-OCT). In order to maintain good visual acuity (VA), focal/grid laser treatment for screened fovea-threatening DME was evaluated based on macular thickness map images produced by SD-OCT. Methods: In 66 diabetic eyes with no visual deterioration, the sensitivity and the specificity of SD-OCT without the use of mydriatics for the detection of fovea-threatening DME were determined. A definite diagnosis of DME was made under mydriasis, using slitlamp biomicroscopy with a contact lens. Eyes with fovea-threatening DME then underwent prophylactic focal/grid laser treatment. The main outcome measures were corrected VA and central macular thickness (CMT). Results: A definitive diagnosis of DME was made in 5 of the 66 eyes, while macular thickening above the 99th percentile was detected in 11 (Cirrus®) or 13 (RS-3000®) eyes by SD-OCT. The focal/grid laser treatment of the 5 eyes with fovea-threatening DME successfully maintained good VA, which was 0.91 ± 0.76 (average: 20/22; 0.04 ± 0.12 logMAR) before treatment and 0.89 ± 0.70 (average: 20/22; 0.05 ± 0.15 logMAR; p = 0.88) 1 year afterwards. The average CMT was stable before and after focal/grid lasering at 302 ± 29 and 329 ± 55 µm, respectively (p = 0.99). Conclusions: The detection of fovea-threatening DME is feasible by SD-OCT without using mydriatics. Prophylactic treatment, such as with focal/grid lasers, was effective in maintaining good VA by avoiding an otherwise highly likely foveal involvement.

Effect of Laser Wavelength on Delivering Appropriate Laser Burns through the Opaque Lens Using a Pattern Scan Laser

Background/Aims: We evaluated the effects of pattern scan laser (PSL) wavelength in delivering appropriate laser burns to the retina of eyes with an opaque lens. Methods: Sixteen shots of 2 × 2 square grids (64 laser spots) were delivered using green (532-nm), yellow (577-nm) and red (647-nm) lasers to the retinas of mice with mild cataract induced by chloral hydrate (400 mg/kg). Three eyes with clear lenses served as controls. One week after laser coagulation, the ratio of appropriate burns, defined as coagulation restricted to the outer half of the retina without retinal or choroidal hemorrhage, was investigated histologically. Results: With the green laser, we confirmed only 3.0 ± 2.0 appropriate burns in eyes with an opaque lens, in contrast to 13.7 ± 4.0 effective burns in eyes with a clear lens. On the other hand, the yellow and red lasers produced 18 ± 5.2 and 13 ± 1.5 appropriate burns, respectively, in eyes with an opaque lens. Conclusion: Although all three PSL wavelengths successfully delivered appropriate burns restricted to the outer half of the retina in eyes with an opaque lens, the longer-wavelength yellow and red lasers were significantly more effective than the green laser. PSL may be a treatment option to accompany anti-vascular endothelial growth factor drug therapy.

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.