Nrf2 Activator RS9 Suppresses Pathological Ocular Angiogenesis and Hyperpermeability.

Purpose: Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. Methods: The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Results: Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-ß, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. Conclusions: These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability.

[Muscle mass measurement by DXA and MRI in non-human primates].

The global population is aging rapidly and, in Japan, the number of elderly has been steadily rising. It is important to shrink the gap between the average lifespan and the number of years people can expect to remain healthy. This links with improving the quality of life for the elderly and reducing social welfare spending. Maintaining motor function is believed to be a key to extending the number of years a person remains healthy, but recent years have seen a rise in locomotive syndrome. Decreases in muscle mass with age, and the deterioration in motor functions leads to sarcopenia. However, there is a dearth of medicines for increasing muscle mass or muscular strength. In this study, we used non-human primates (NHPs), which have similar anatomical features to humans and have advanced functional differentiation between the fore- and hindlimbs, to examine a highly accurate method of measuring muscle mass using Magnetic Resonance Imaging (MRI) and compared it to Dual Energy X-ray Absorptiometry (DXA) usually used in clinical settings. The results showed that both MRI and DXA provided high repeatability. Furthermore, correlation analysis between the amount of excised muscle for measurement and the results from MRI and DXA showed a high correlation at all sites examined, with the correlation coefficient higher for MRI than for DXA. We expect that the establishment of a highly accurate method for measurement of muscle mass using MRI and DXA will give impetus to the development of drugs that target muscle mass.

Healing of two-wall intra-bony defects treated with a novel EMD-liquid-A pre-clinical study in monkeys.

AIM: To investigate the effect of a novel enamel matrix derivative formulation (EMD-liquid or Osteogain) combined with an absorbable collagen sponge (ACS) on periodontal wound healing in intra-bony defects in monkeys. MATERIALS AND METHODS: Chronic two-wall intra-bony defects were created at the distal aspect of eight teeth in three monkeys (Macaca fascicularis). The 24 defects were randomly assigned to one of the following treatments: (i) open flap debridement (OFD) + ACS alone, (ii) OFD + Emdogain + ACS (Emdogain/ACS), (iii) OFD + Osteogain + ACS (Osteogain/ACS) or (iv) OFD alone. At 4 months, the animals were euthanized for histologic evaluation. RESULTS: Osteogain/ACS resulted in more consistent formation of cementum, periodontal ligament and bone with limited epithelial proliferation compared to OFD alone, Emdogain/ACS and OFD + ACS. Among the four treatment groups, the Osteogain/ACS group demonstrated the highest amount of regenerated tissues. However, complete periodontal regeneration was not observed in any of the defects in the four groups. CONCLUSIONS: The present findings indicate that in two-wall intra-bony defects, reconstructive surgery with Osteogain/ACS appears to be a promising novel approach for facilitating periodontal wound healing/regeneration, thus warranting further clinical testing.

Movement of retinal vessels toward the optic nerve head after increasing intraocular pressure in monkey eyes with experimental glaucoma.

A shift or displacement of the retinal blood vessels (RBVs) with neuroretinal rim thinning indicates the progression of glaucomatous optic neuropathy. In chronic open angle glaucoma, individuals with RBV positional shifts exhibit more rapid visual field loss than those without RBV shifts. The retinal vessels reportedly move onto the optic nerve head (ONH) in response to glaucoma damage, suggesting that RBVs are pulled toward the ONH in response to increased cupping. Whether this phenomenon only applies to RVBs located in the vicinity or inside the ONH or, more generally, to RBVs also located far from the ONH, however, is unclear. The aim of this study was to evaluate the movement of RBVs located relatively far from the ONH edge after increasing intraocular pressure (IOP) in an experimental monkey model of glaucoma. Fundus photographs were obtained in 17 monkeys. High IOP was induced in the monkeys by laser photocoagulation burns applied uniformly with 360° irradiation around the trabecular meshwork of the left eye. The right eye was left intact and used as a non-treated control. Considering the circadian rhythm of IOP, it was measured in both eyes of each animal at around the same time-points. Then, fundus photographs were obtained. Using Image J image analysis software, an examiner (N.E.) measured the fundus photographs at two time-points, i.e. before laser treatment (time 1) and the last fundus photography after IOP elevation (time 2). The following parameters were measured (in pixels): 1) vertical diameter of the ONH (DD), 2) distance from the ONH edge to the first bifurcation point of the superior branch of the central retinal vein (UV), 3) distance from the ONH edge to the first bifurcation point of the inferior branch of the central retinal vein (LV), 4) ONH area, and 5) surface area of the cup of the ONH. We calculated the ratios of UV to DD (UV/DD), LV to DD (LV/DD), and the cup area to disc area ratio (C/D). The mean UV/DD at time 1 (0.656 ± 0.233) was decreased at time 2 (0.542 ± 0.192) (p < 0.01), and the mean LV/DD at time 1 (0.642 ± 0.151) was decreased at time 2 (0.534 ± 0.171) (p < 0.01). The mean C/D at time 1 (0.303 ± 0.035) was increased at time 2 (0.556 ± 0.110) (p < 0.01). The mean IOP at time 1 was 19.8 ± 2.5 and that at time 2 was 54.2 ± 15.8. The amount and rate of the change in LV/DD and C/D between time 1 and time 2 were significantly correlated (r = -0.654 and -0.536, p = 0.004 and 0.026, respectively). Therefore, in an experimental monkey model of glaucoma, RBVs located relatively far from the ONH were pulled toward the ONH as cupping increased.

Relationship between Elevated Intraocular Pressure and Divided Peripapillary Sector Retinal Nerve Fiber Layer Thickness in a Cynomolgus Monkey Laser-Induced Ocular Hypertension Model.

AIMS: We investigated the relationship between elevated intraocular pressure (IOP) and changes in global and peripapillary sector retinal nerve fiber layer (RNFL) thickness around the optic nerve head (ONH) in the laser-induced ocular hypertension monkey model. METHODS: To induce high IOP, green laser photocoagulation burns were applied around the trabecular meshwork of 1 eye from each of 12 cynomolgus monkeys. The animals had been acclimated to IOP measurement under conscious conditions for more than 2 months, and IOP was chronologically measured. RNFL thickness was measured for 6 peripapillary sectors and global area using spectral-domain optical coherence tomography. RESULTS: After model induction, marked IOP elevation and enlarged optic disk cupping were observed. Thinning of the RNFL associated with elevated IOP was observed around the ONH from 6 until 9 weeks after laser treatment, and the degree of reduction in RNFL thickness varied between the peripapillary sectors. Correlations between cumulative IOP elevation and RNFL thickness reduction were statistically significant for the temporal-superior (p = 0.024), nasal-inferior (p = 0.044), and temporal (p = 0.049) sectors, and global RNFL (p = 0.018). CONCLUSIONS: These results suggest that this model reflected the pathology of clinical glaucoma in terms of the specific pattern of RNFL thinning around the ONH.

Effects of EMD liquid (Osteogain) on periodontal healing in class III furcation defects in monkeys.

AIM: To evaluate the effect of a novel liquid carrier system of enamel matrix derivative (Osteogain) soaked on an absorbable collagen sponge (ACS) upon periodontal wound healing/regeneration in furcation defects in monkeys. MATERIALS AND METHODS: The stability of the conventional enamel matrix derivative (Emdogain) and Osteogain adsorbed onto ACS was evaluated by ELISA. Chronic class III furcation defects were created at teeth 36, 37, 46, 47 in three monkeys (Macaca fascicularis). The 12 defects were assigned to one of the following treatments: (1) open flap debridement (OFD) + ACS, (2) OFD+Emdogain/ACS, (3) OFD+Osteogain/ACS, and (4) OFD alone. At 16 weeks following reconstructive surgery, the animals were killed for histological evaluation. RESULTS: A 20-60% significantly higher amount of total adsorbed amelogenin was found for ACS-loaded Osteogain when compared to Emdogain. The histomorphometric analysis revealed that both approaches (OFD + Emdogain/ACS and OFD + Osteogain/ACS) resulted in higher amounts of connective tissue attachment and bone formation compared to treatment with OFD + ACS and OFD alone. Furthermore, OFD + Osteogain/ACS group showed higher new attachment formation, cementum, and new bone area. CONCLUSIONS: Within their limits, the present data indicate that Osteogain possesses favourable physicochemical properties facilitating adsorption of amelogenin on ACS and may additionally enhance periodontal wound healing/regeneration when compared to Emdogain.

Joint Degradation in a Monkey Model of Collagen-Induced Arthritis: Role of Cathepsin K Based on Biochemical Markers and Histological Evaluation.

The role of cathepsin K in joint degradation in a model of collagen-induced arthritis (CIA) in cynomolgus monkey was examined using biochemical markers and histology. Joint swelling, urinary C-telopeptide of type II collagen (CTX-II), deoxypyridinoline (DPD), and N- and C-telopeptides of type I collagen (NTX and CTX-I, resp.) were analyzed. Immunohistochemistry of type II collagen, cathepsin K, and CTX-II were performed using joints. Joint swelling reached peak on day 42 and continued at this level. The CTX-II level peaked on day 28 and declined thereafter, while CTX-I, NTX, and DPD reached plateau on day 43. Joint swelling was positively correlated with CTX-II increases on days 20 and 42/43, with increases in CTX-I and NTX/Cr on days 42/43 and 84, and with DPD increases throughout the study period. Intense cathepsin K staining was observed in osteoclasts and in articular cartilage and synovial tissue in arthritic joints. CTX-II was present in the superficial layer of articular cartilage in CIA monkeys. Evidence from biochemical markers suggests that matrix degradation in the CIA model starts with degradation of cartilage, rather than bone resorption. Cathepsin K expressed in osteoclasts, articular cartilage, and synovial tissue may contribute to degradation of cartilage.

Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-Induced Arthritis in Cynomolgus Monkeys.

Interleukin-6 (IL-6) is a potent mediator of inflammatory and immune responses, and a validated target for therapeutic intervention of inflammatory diseases. Previous studies have shown that SL1026, a slow off-rate modified aptamer (SOMAmer) antagonist of IL-6, neutralizes IL-6 signaling in vitro. In the present study, we show that SL1026 delays the onset and reduces the severity of rheumatoid symptoms in a collagen-induced arthritis model in cynomolgus monkeys. SL1026 (1 and 10 mg/kg), administered q.i.d., delayed the progression of arthritis and the concomitant increase in serum IL-6 levels compared to the untreated control group. Furthermore, SL1026 inhibited IL-6-induced STAT3 phosphorylation ex vivo in T lymphocytes from human blood and IL-6-induced C-reactive protein and serum amyloid A production in human primary hepatocytes. Importantly, SOMAmer treatment did not elicit an immune response, as evidenced by the absence of anti-SOMAmer antibodies in plasma of treated monkeys. These results demonstrate that SOMAmer antagonists of IL-6 may be attractive agents for the treatment of IL-6-mediated diseases, including rheumatoid arthritis.

Muscle wasting associated with pathologic change is a risk factor for the exacerbation of joint swelling in collagen-induced arthritis in cynomolgus monkeys.

BACKGROUND: Not only joint destruction but also muscle wasting due to rheumatoid cachexia has been problem in terms of quality of life of patients with rheumatoid arthritis (RA). In the present study, we performed histopathological examination and assessed relationships between characteristic parameters relating to muscle and joint swelling in a collagen-induced arthritis (CIA) model using cynomolgus monkeys (CMs). METHODS: Female CMs were used and CIA was induced by twice immunizations using bovine type II collagen with Freund's complete adjuvant. Arthritis level was evaluated from the degree of swelling at the peripheral joints of the fore and hind limbs. Food consumption, body weight, and serum biochemical parameters were measured sequentially. Five or 6 animals per time point were sacrificed at 2, 3, 5 and 9 weeks after the first immunization to obtain quadriceps femoris specimens for histopathology. Pimonidazole hydrochloride was intravenously administered to determine tissue hypoxia in skeletal muscle. RESULTS: Gradual joint swelling was observed and the maximum arthritis score was noted at Week 5. In histopathology, necrosis of muscle fiber in the quadriceps femoris was observed only at Week 2 and the most significant findings such as degeneration, atrophy, and regeneration of muscle fiber were mainly observed at Week 5. Food consumption was decreased up to Week 4 but recovered thereafter. Body weight decreased up to Week 5 and did not completely recover thereafter. A biphasic increase in serum cortisol was also observed at Weeks 2 and 5. Histopathology showed that muscle lesions were mainly composed of degeneration and atrophy of the muscle fibers, and ATPase staining revealed that the changes were more pronounced in type II muscle fiber than type I muscle fiber. In the pimonidazole experiment, mosaic pattern in skeletal muscle was demonstrated in the intact animal, but not the CIA animal. Increased arthritis score was accompanied by a decrease in serum creatinine, a marker that reflects muscle mass. CONCLUSIONS: Muscle wasting might exacerbate joint swelling in a collagen-induced arthritis model of cynomolgus monkeys.

Tocilizumab, a humanized anti-interleukin-6 receptor antibody, improved anemia in monkey arthritis by suppressing IL-6-induced hepcidin production.

We have reported that serum IL-6 level was related with the degree of anemia in monkey collagen-induced arthritis (CIA). In this study, we examined whether IL-6 blockade ameliorated an anemia in monkey CIA. CIA was induced by twice immunization of bovine type II collagen with adjuvant. When anemia became evident, anti-IL-6 receptor antibody, tocilizumab was intravenously injected once a week for 4 weeks. Controls received PBS in a same manner. Hematological and biochemical parameters were measured regularly and serum hepcidin-25 levels were measured by SELDI-TOF mass spectrometry. Moreover, hepcidin mRNA induction in Hep3B cells by serum from arthritic monkeys was examined by real-time PCR. Administration of tocilizumab rapidly decreased CRP levels and improved iron-deficient anemia within 1 week. Tocilizumab induced rapid but transient reduction in serum hepcidin-25. Hepcidin mRNA expression was more potently induced by serum from arthritic monkey and this was inhibited by the addition of tocilizumab. Blockade of IL-6 signaling rapidly improved anemia in monkey arthritis via the inhibition of IL-6-induced hepcidin production.

Extracellular high mobility group box chromosomal protein 1 is a coupling factor for hypoxia and inflammation in arthritis.

OBJECTIVE: Tissue hypoxia is closely associated with arthritis pathogenesis, and extracellular high mobility group box chromosomal protein 1 (HMGB-1) released from injured cells also has a role in arthritis development. This study was thus undertaken to investigate the hypothesis that extracellular HMGB-1 may be a coupling factor between hypoxia and inflammation in arthritis. METHODS: Concentrations of tumor necrosis factor alpha, interleukin-6, vascular endothelial growth factor, lactic acid, lactate dehydrogenase, and HMGB-1 were measured in synovial fluid (SF) samples from patients with inflammatory arthropathy (rheumatoid arthritis and pseudogout) and patients with noninflammatory arthropathy (osteoarthritis). The localization of tissue hypoxia and HMGB-1 was also examined in animal models of collagen-induced arthritis (CIA). In cell-based experiments, the effects of hypoxia on HMGB-1 release and its associated cellular events (i.e., protein distribution and cell viability) were studied. RESULTS: In SF samples from patients with HMGB-1-associated inflammatory arthropathy (i.e., samples with HMGB-1 levels >2 SD above the mean level in samples from patients with noninflammatory arthropathy), concentrations of HMGB-1 were significantly correlated with those of lactic acid, a marker of tissue hypoxia. In CIA models in which the pathologic phenotype could be attenuated by HMGB-1 neutralization, colocalization of HMGB-1 with tissue hypoxia in arthritis lesions was also observed. In cell-based experiments, hypoxia induced significantly increased levels of extracellular HMGB-1 by the cellular processes of secretion and/or apoptosis-associated release, which was much more prominent than the protein release in necrotic cell injury potentiated by oxidative stress. CONCLUSION: These findings indicate that tissue hypoxia and its resultant extracellular HMGB-1 might play an important role in the development of arthritis.