Three-year outcome of photodynamic therapy combined with VEGF inhibitor for pachychoroid neovasculopathy.

BACKGROUND: Long-term results of photodynamic therapy (PDT) combined with vascular endothelial growth factor (VEGF) inhibitors for pachychoroid neovasculopathy (PNV) are not yet clear. METHODS: This study is a retrospective, observational case series. We retrospectively examined untreated PNV cases (22 cases, 22 eyes, mean age of 71.0 years) who underwent PDT therapy in combination with VEGF inhibitors followed by additional treatments with pro re nata protocol. Visual acuity, number of treatments, and time to recurrence were examined. In addition, foveal choroidal thickness and choroidal vascularity index (CVI) were evaluated in 13 of 22 patients who were followed up with SpectralisOCTR from baseline. RESULTS: Fifteen (68%) cases had polyps at baseline. LogMAR visual acuity averaged 0.24 ± 0.20 (range, - 0.079 to 0.82) at baseline and significantly improved after 1, 2, and 3 years (p = 0. 004, 0.0003, 0.002, respectively). Fourteen patients (64%) recurred, with an average time to recurrence of 1.8 ± 0.9 years. Foveal choroidal thickness decreased significantly after 1 year (average from 326 µm to 263 µm) and remained unchanged up to 3 years (255 µm). CVI also decreased after 1 year (average from 0.62 to 0.61) and remained unchanged until 3 years later (0.60). CONCLUSIONS: We examined the 3-year course of PDT in combination with the VEGF inhibitor for untreated PNV. Visual acuity was improved, foveal choroidal thickness and CVI were decreased after 3 years.

Intravitreal anti-vascular endothelial growth factor and combined photodynamic therapy for pachychoroid neovasculopathy: long-term treatment outcomes.

PURPOSE: To examine the long-term visual outcomes after initial treatment with combined photodynamic therapy (PDT) or aflibercept treat-and-extend (TAE) monotherapy in patients with pachychoroid neovasculopathy (PNV). METHODS: Patients diagnosed with PNV, initially treated with PDT combined with anti-vascular endothelial growth factor (VEGF) or intravitreal aflibercept (IVA) monotherapy in the TAE protocol and followed up for at least 6 months, were included in the study. Medical records were retrospectively reviewed. Survival analysis was performed, in which deterioration in logMAR visual acuity by 0.1 or 0.3 is defined as "death." The annual number of treatments was also analyzed. Sub-analysis was performed on 33 patients diagnosed with PNV without polypoidal lesions. RESULTS: This study included 46 patients (23 in the initial combined PDT group and 23 in the IVA TAE group). Mean age, sex, mean baseline logMAR visual acuity, or duration of observation (3.6 ± 3.2 years vs. 3.1 ± 1.9 years) in both groups were comparable. As for visual outcome, no significant differences were found in survival analysis based on worsening of 0.1 or 0.3 logMAR (3-year survival; 26% vs. 26%, 91% vs. 90%, respectively). Meanwhile, the additional number of anti-VEGF injections per year was significantly lower in the initial combined PDT group than in the IVA TAE group (1.0 ± 1.3 vs. 4.1 ± 1.5, p < 0.0001). No significant differences were found in the number of additional PDTs per year (0.07 ± 0.20 vs. 0.02 ± 0.09, p = 0.27). Similar results were found in a sub-analysis of 33 patients without polyps. CONCLUSION: In the treatment of PNV, regardless of the presence of polyps, the long-term visual outcomes were similar between the initial combined PDT and IVA TAE monotherapy. However, the annual number of anti-VEGF injections was lower in the initial combined PDT group than in the aflibercept TAE group, whereas that of PDT was comparable.

Immunogammopathy Maculopathy Secondary to Waldenström's Macroglobulinemia Complicated With Diabetic Retinopathy: A Case Report and Literature Review.

In Waldenström's Macroglobulinemia (WM), increased immunoglobulin M causes various signs and symptoms. It sometimes presents with macular edema. A 65-year-old WM patient with a five-year history of diabetes mellitus was evaluated for ocular complications. Fundus examination and optical coherence tomography showed retinal changes consistent with non-proliferative diabetic retinopathy and foveal detachment with intraretinal cysts in the right eye, suggesting diabetic macular edema. However, on fluorescein angiography, there was no leakage over the area of foveal detachment, which led to the diagnosis of immunogammopathy maculopathy secondary to WM for macular edema and foveal detachment. The patient's ocular manifestation remained unchanged through a follow-up period of 11 months without therapeutic interventions. Immunogammopathy maculopathy, a rare ocular manifestation of monoclonal gammopathy, demands differentiation from other causes of macular edema in WM patients. The present case highlights the importance of fluorescein angiography, or silent macula, in diabetic patients to distinguish immunogammopathy maculopathy from diabetic macular edema.

NGAL is a Novel Target in Hypertension by Modulating the NCC-Mediated Renal Na Balance.

BACKGROUND: The expression of NGAL/lcn2 (neutrophil gelatinase-associated lipocalin) is directly modulated by mineralocorticoid receptor activation but its role in blood pressure control is unclear. METHODS: a potential relationship between NGAL plasma levels, systolic blood pressure and urinary Na excretion was assessed in the STANISLAS cohort. The specific role of NGAL/lcn2 in salt-sensitive hypertension was studied using lcn2-knockout mice (lcn2 KO) fed with low-Na diet (0Na). RESULTS: we show that NGAL plasma levels positively correlate with systolic blood pressure, whereas they negatively correlate with urinary Na excretion in subjects of the STANISLAS cohort. Prolonged feeding of lcn2 KO mice with a 0Na diet induced lower systolic blood pressure than that of the control group (wildtype), suggesting a role for NGAL/lcn2 in Na-balance homeostasis. Short-term or prolonged 0Na increased Na-Cl cotransporter (NCC) phosphorylation in the cortex of wildtype mice, which was prevented in lcn2 KO mice. Recombinant mouse lcn2 injections in lcn2 KO mice induced NCC phosphorylation in the kidney cortex, associated with decreased urinary Na excretion. Ex vivo experiments using kidney slices from lcn2 KO mice showed increased NCC phosphorylation by recombinant murine lcn2. In addition, recombinant murine lcn2 induced activation of CamK2ß (calcium/calmodulin-dependent protein kinase II ß subunit) phosphorylation in lcn2 KO mice and in kidney slices, providing an underlying mechanism involved in lcn2-induced NCC phosphorylation. Indeed, the inhibition of CamK2ß prevented NCC phosphorylation induced by recombinant lcn2 in kidney slices. CONCLUSIONS: we highlight a novel role of NGAL/lcn2 as a modulator of the activity of the renal sodium transporter NCC affecting salt-sensitive blood pressure.

Rapidly Changing Serous Detachment During BRAF and MEK Inhibitor Therapy.

We report a case of rapidly changing serous retinal detachment (SRD) during melanoma therapy with a combination of encorafenib, a serine/threonine-protein kinase B-Raf (BRAF) inhibitor, and binimetinib, a mitogen-activated protein kinase (MEK) inhibitor. A 50-year-old woman with metastatic melanoma presented with a sudden visual blur. She had been treated with encorafenib (450 mg every morning) and binimetinib (45 mg every 12 hours) after surgery for four months. Ophthalmological examination revealed bilateral SRD, but it was completely resolved after two hours. Visual acuity was normal in each eye. Encorafenib and binimetinib were continued. Shallow SRD appeared again five months later, but it resolved in two months. MEKAR typically occurs shortly after the start of an administration, and its development after several months was very little known. Continued examination for ophthalmic events should be considered.

Pharmacokinetics and safety after once and twice a day doses of meclizine hydrochloride administered to children with achondroplasia.

Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. We identified that meclizine hydrochloride inhibited FGFR3 signaling in various chondrocytic cells and promoted longitudinal bone growth in mouse model of ACH. Meclizine has safely been used for more than 50 years, but it lacks the safety data for repeated administration and pharmacokinetics (PK) when administered to children. We performed a phase Ia study to evaluate the PK and safety of meclizine administered orally to ACH children. Twelve ACH children aged from 5 to younger than 11 years were recruited, and the first 6 subjects received once a day of meclizine in the fasted condition, subsequent 6 subjects received twice a day of meclizine in the fed condition. Meclizine was well tolerated in ACH children with no serious adverse events. The mean Cmax, Tmax, AUC0-24h, t1/2 during 24 hours in the fasted condition were 130 ng/mL, 1.7 hours, 761 ng·h/mL, and 8.5 hours respectively. The simulation of repeated administration of meclizine for 14 days demonstrated that plasma concentration apparently reached steady state around 10 days after the first dose both at once a day and twice a day administration. The AUC0-10h of the fasting and fed condition were 504 ng·h/mL and 813 ng·h/mL, respectively, indicating exposure of meclizine increased with the diet. Although higher drug exposure was confirmed in ACH children compared to adults, a single administration of meclizine seemed to be well tolerated.

Podocyte Injury Augments Intrarenal Angiotensin II Generation and Sodium Retention in a Megalin-Dependent Manner.

We have previously shown that podocyte injury increases the glomerular filtration of liver-derived Agt (angiotensinogen) and the generation of intrarenal Ang II (angiotensin II) and that the filtered Agt is reabsorbed by proximal tubules in a manner dependent on megalin. In the present study, we aimed to study the role of megalin in the generation of renal Ang II and sodium handling during nephrotic syndrome. We generated proximal tubule-specific megalin KO (knockout) mice and crossed these animals with NEP25 mice, in which podocyte-specific injury can be induced by injection of the immunotoxin LMB2. Without podocyte injury, renal Agt staining was markedly diminished and urinary Agt increased in KO mice. However, renal Ang II was similar between KO and control mice on average: 117 (95% CI, 101-134) versus 101 (95% CI, 68-133) fmol/g tissue. We next tested the effect of megalin KO on intrarenal Ang II generation with podocyte injury. Control NEP25 mice showed markedly increased renal Agt staining and renal Ang II levels: 450 (336-565) fmol/g tissue. Megalin KO/NEP25 mice showed markedly diminished Agt reabsorption and attenuated renal Ang II: 199 (156-242) fmol/g tissue (P<0.001). Compared with control NEP25 mice, megalin KO/NEP25 mice excreted 5-fold more sodium in the urine. Western blot analysis showed that megalin KO decreased NHE3 and the cleaved α and γ forms of Epithelial Na Channel. These data indicate that Agt reabsorbed by proximal tubules via megalin in nephrotic syndrome is converted to Ang II, which may contribute to sodium retention and edema formation by activating NHE3 and Epithelial Na Channel.

Aberrant DNA methylation of pregnane X receptor underlies metabolic gene alterations in the diabetic kidney.

Epigenetic abnormalities have been suggested to mediate metabolic memory observed in diabetic complications. We have shown that epigenetic alterations may induce persistent phenotypic changes in the proximal tubules of the diabetic kidneys. In this study, we show that pregnane X receptor (PXR), a xenobiotic nuclear receptor, is epigenetically altered and upregulated and may have a possible function in the diabetic kidney. PXR has been shown to play a critical role in metabolic changes in obesity and diabetes; however, its distribution and function in the kidney are unknown. In the normal kidney, Pxr was selectively expressed in the proximal tubular cells with demethylation in the promoter DNA. In db/db mice, significant increases in Pxr mRNA, further demethylation of DNA, and stimulatory histone marks in the promoter were observed. Epigenetic changes are likely to play a causative role in PXR induction, since a DNA methyltransferase inhibitor increased PXR mRNA in cultured human proximal tubular cells. Administration of a PXR agonist increased mRNA levels of solute carrier organic anion transporter family member 2B1 ( Slco2b1), a xenobiotic transporter; response gene to complement 32 ( Rgc32), a molecule known to exert fibrotic effects in the kidney; and phosphoenolpyruvate carboxykinase 1 ( Pck1), a gluconeogenic enzyme in the kidney. The expressions of these genes were inhibited by PXR small interfering RNA in cultured proximal tubular cells. Increased mRNA levels of Slco2b1, Rgc32, and Pck1 were also observed in the kidney of db/db mice. These data indicate that PXR is upregulated in the diabetic kidney with aberrant epigenetic modifications and may modulate the course of diabetic kidney disease through the activation of these genes.

Oxidative stress causes mineralocorticoid receptor activation in rat cardiomyocytes: role of small GTPase Rac1.

Overactivation of the mineralocorticoid receptor signaling is implicated in cardiovascular disease, including hypertensive heart disease. Oxidative stress is suggested to augment mineralocorticoid receptor signal transduction, but the precise mechanisms remain unclear. Mineralocorticoid receptor activity is regulated by multiple factors, in addition to plasma ligand levels. We previously identified Rac1 GTPase as a modulator of mineralocorticoid receptor activity. Here we show that oxidative stress induces mineralocorticoid receptor activation in a ligand-independent, Rac1-depenent manner in cardiomyocytes. Oxidant stress was induced in rat cultured cardiomyocytes (H9c2) by l-buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO depleted intracellular glutathione and concomitantly increased reactive oxygen species (199%; P<0.01). BSO significantly enhanced the corticosterone-induced, mineralocorticoid receptor-dependent luciferase reporter activity (186%; P<0.01) and basal luciferase activity without ligand stimulation. These effects were inhibited by the antioxidant N-acetylcysteine. The ligand independency of BSO action was indicated using a mutant mineralocorticoid receptor that does not bind ligands. With this mutant mineralocorticoid receptor, BSO-evoked mineralocorticoid receptor activation remained intact, whereas ligand-induced mineralocorticoid receptor activation was abolished. We next examined the involvement of Rac1. BSO increased active Rac1 in a redox-dependent fashion, and Rac inhibition suppressed the enhancing effect of BSO. Constitutively active Rac1, indeed, potentiated mineralocorticoid receptor transactivation. Furthermore, mineralocorticoid receptor transactivation by BSO was accompanied by enhanced nuclear accumulation of mineralocorticoid receptor. We conclude that alteration of redox state modulates mineralocorticoid receptor-dependent transcriptional activity via Rac1 in the heart. This redox-sensitive, ligand-independent mineralocorticoid receptor activation may contribute to the processes by which oxidant stress promotes cardiac injury.

Heparin induces apoptosis through suppression of AKt in oral squamous cell carcinoma cells.

BACKGROUND: Heparin is a polysulfated glycosaminoglycan that has been shown to have antiproliferative and apoptotic effects in addition to its anticoagulant effects. MATERIALS AND METHODS: The present work investigated the effects of unfractioned heparin (UFH) on cell growth and apoptosis in four oral squamous cell carcinoma (SCC) cell lines and the mechanism(s) underlying its actions using MTT assay, Annexin-V-FITC and Western blotting. RESULTS: Treatment with UFH resulted in significant reduction in cell viability and increase in apoptosis in three of the four tested cell lines. Further, such treatment resulted in a significant decrease in phosphorylated AKt, and consequently led to activation of the mitochondrial pathway in heparin-sensitive cells. Moreover, pretreatment with UFH significantly increased the apoptosis induced by cisplatin. CONCLUSION: These findings indicate that heparin induces apoptosis through suppression of AKt, and suggest a potential utility of heparin for development of less toxic chemotherapy in treatment of oral SCC.

Changes in serum and exudate creatine phosphokinase concentrations as an indicator of deep tissue injury: a pilot study.

Deep tissue injury (DTI) is difficult to detect in the early phase. Creatine phosphokinase (CPK) as a muscle enzyme could represent a promising indicator of DTI. However, serum CPK levels reflect the systemic condition rather than the local wound environment. Wound exudates can be indicative of the local wound environment. This study aimed to investigate the usefulness of CPK levels in wound exudates as an indicator of DTI. Rats were divided into control, 6 hours 10-kg and 6 hours 20-kg loading groups. Serum samples were obtained before wounding, and at 8 and 12 hours, and 1, 2 and 3 days after wounding, while exudate samples were obtained on days 2 and 3. Serum CPK levels were markedly increased in the 10-kg and 20-kg groups at 8 and 12 hours after loading compared with the baseline value and control group, but decreased to the normal level on day 1. In both loading groups, exudate CPK levels were high on day 2 and decreased on day 3. Muscle necrosis was more severe in the 20-kg group than in the 10-kg group by histological examination. This is the first study to indicate the potential of CPK in wound exudates as an indicator of DTI.

Endoplasmic reticulum stress induces Wfs1 gene expression in pancreatic beta-cells via transcriptional activation.

OBJECTIVE: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic beta-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses. DESIGN AND METHODS: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic beta-cell-derived MIN6 cells, and Akita mouse-derived Ins2 (96Y/Y) insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted. RESULT: Islet beta-cells were the major site of Wfs1 expression. This expression was also found in delta-cells, but not in alpha-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 (96Y/Y) insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 (96Y/Y) cells than Ins2 (wild/wild) cells. CONCLUSION: Wfs1 expression, which is localized to beta- and delta-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.

Interferon-{beta} inhibits bleomycin-induced lung fibrosis by decreasing transforming growth factor-{beta} and thrombospondin.

Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-beta is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-beta did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-beta is a potent inhibitor of fibrogenesis, beneficial effects of IFN-beta have been expected. We therefore attempted to determine effects of IFN-beta and investigated the mechanism of action of IFN-beta in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-beta for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-beta. Tissue distribution and amounts of TGF-beta1 and thrombospondin (TSP)-1/2 were analyzed. IFN-beta attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-beta1 in epithelial cells and extracellular matrix was attenuated by IFN-beta. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-beta is inhibition of TGF-beta and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.