Effect of the combination of cationic lipid and phospholipid on gene-knockdown using siRNA lipoplexes in breast tumor cells and mouse lungs.

Previously, using three types of cationic lipids, the effect of phospholipids in liposomal formulations on gene-knockdown efficacy was determined after in vitro and in vivo transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) containing various cationic lipids and phospholipids. In the present study, six other types of cationic lipids, namely N,N-dimethyl-N-tetradecyltetradecan-1-aminium bromide, N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 2-[bis{2-(tetradecanoyloxy)ethyl}amino]-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-6-14), 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride (DOTMA), 1,2-distearoyl-3-trimethylammonium-propane chloride (DSTAP) and 1,2-dioleoyl-3-dimethylammonium-propane were selected, and the effect of phospholipids in liposomal formulations containing each cationic lipid on gene-knockdown was evaluated. A total of 30 types of cationic liposomes composed of each cationic lipid with phosphatidylethanolamine containing unsaturated or saturated diacyl chains (C14, C16 or C18) were prepared. Regardless of the type of cationic lipid, the inclusion of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the liposomal formulations resulted in injectable size of siRNA lipoplexes after mixing of siRNA and cationic liposomes. Transfection of their lipoplexes with luciferase (Luc) siRNA into human breast cancer MCF-7-Luc cells stably expressing Luc led to a strong knockdown of Luc. Furthermore, the systemic injection of siRNA lipoplexes composed of DC-1-16, DC-6-14, DOTMA or DSTAP with DOPE resulted in siRNA accumulation in the lungs. Significant gene-knockdown was observed in the lungs of mice following the systemic injection of siRNA lipoplexes containing DC-1-16 and DOPE. Cationic liposomes composed of DC-1-16 and DOPE serve as potential carriers for in vitro and in vivo siRNA transfection.

The usefulness of the retinal sensitivity measurement with a microperimetry for predicting the visual prognosis of branch retinal vein occlusion with macular edema.

PURPOSE: To evaluate the usefulness of the retinal sensitivity in branch retinal vein occlusion (BVO) with macular edema (ME) following the anti-vascular endothelial growth factor (anti-VEGF) treatment. METHODS: Best-corrected visual acuity (BCVA), microperimetry, and optical coherence tomography (OCT) measurements were carried out in 20 patients with BVO with ME, at baseline and 1 month after the anti-VEGF treatment. The relationships among BCVA, mean retinal sensitivity (MS), macular volume (MV), central retinal thickness (CRT), integrity of ellipsoid zone (EZ), mean retinal sensitivity in the most affected quadrant (qMS), and macular volume in the most affected quadrant (qMV) were investigated. In addition, the relationships among the change in BCVA at 1 month (ΔBCVA1m), mean sensitivity in the most affected quadrant at 1 month (ΔqMS1m), MV in the most affected quadrant at 1 month (ΔqMV1m), and CRT at 1 month (ΔCRT1m) were analyzed. The optimal model for BCVA at 3 months after the treatment (BCVA3m) was identified. RESULTS: There was not a significant difference in BCVA (paired Wilcoxon test, p = 0.058) between at baseline and after the treatment, but there were significant differences in MS, MV, CRT, qMS, and qMV (p < 0.05). There was a significant relationship between ΔqMS1m and ΔMV1m, ΔCRT1m, and ΔqMV1m, respectively. ΔMS1m or ΔqMS1m and BCVA at baseline and ΔBCVA1m were selected as explanatory variables in the optimal model for BCVA3m. CONCLUSION: Retinal sensitivity was related to retinal structure, whereas this was not the case with BCVA. In addition, retinal sensitivity was useful to predict BCVA after anti-VEGF therapy.

The regulatory mechanism involved in the prostaglandin E2 disposition in carbon tetrachloride-induced liver injury.

Prostaglandin E2 (PGE2) exhibits hepatoprotective effects against various types of liver injury. However, there is little information on the disposition of endogenous PGE2 during liver injury. In the present study, we attempted to elucidate the mechanism involved in regulating PGE2 distribution during liver injury. Carbon tetrachloride (CCl4) was used to establish a liver injury mouse model. PGE2 was measured by LC-MS/MS. The plasma and hepatic PGE2 levels were significantly increased at 6 to 48 h after CCl4 treatment. The ratio of plasma levels of 13,14-dihydro-15-ketoPGE2 (PGEM), a major PGE2 metabolite, to PGE2 decreased significantly after CCl4 treatment. PGE2 synthesis and expression of enzymes related to PGE2 production were not induced, while the activity and mRNA expression of 15-prostaglandin dehydrogenase (15-PGDH/Hpgd), a major enzyme for PGE2 inactivation, decreased significantly in the liver of CCl4-treated mice compared to that of vehicle-treated control. The plasma and hepatic PGE2 levels were negatively correlated with the hepatic mRNA expression levels of Hpgd. Although the mRNA expression of organic anion transporting polypeptide 2A1 (OATP2A1/Slco2a1), a major PGE2 transporter, was upregulated, other hepatic OATPs decreased significantly at 24 h after CCl4 treatment. Immunohistochemical analysis indicated that 15-PGDH was mainly expressed in endothelial cells and that OATP2A1 was expressed at least in endothelial cells and Kupffer cells in the liver. These results suggest that the decreased 15-PGDH expression in hepatic endothelial cells is the principal mechanism for the increase in hepatic and plasma PGE2 levels due to the CCl4-induced liver injury.

Dietary n-3 Fatty Acid, α-Tocopherol, Zinc, vitamin D, vitamin C, and ß-carotene are Associated with Age-Related Macular Degeneration in Japan.

This case-control study reports the association between nutrient intake and neovascular age-related macular degeneration (AMD) in Japan. The nutrient intake of 161 neovascular AMD cases from two university hospitals and 369 population-based control subjects from a cohort study was assessed using a brief-type self-administered questionnaire on diet history, which required respondent recall of the usual intake of 58 foods during the preceding month. Energy-adjusted nutrient intake values were compared between the groups. Logistic regression analysis was used to estimate odds ratios (ORs) and 95% CIs adjusted for smoking history, age, sex, chronic disease history, supplement use, and alcohol consumption. Logistic regression analysis demonstrated that low intakes of n-3 fatty acid, α-tocopherol, zinc, vitamin D, vitamin C, and ß-carotene were associated with neovascular AMD (Trend P < 0.0001 for n-3 fatty acid, Trend P < 0.0001 for α-tocopherol, Trend P < 0.0001 for zinc, Trend P = 0.002 for vitamin D, Trend P = 0.04 for vitamin C, Trend P = 0.0004 for ß-carotene). There was no association with retinol or cryptoxanthin intake and neovascular AMD (P = 0.67, 0.06).

Risk Factors for Age-Related Macular Degeneration in an Elderly Japanese Population: The Hatoyama Study.

PURPOSE: To estimate the risk factors of AMD in an elderly Japanese population from a suburban area north of metropolitan Tokyo. METHODS: The Hatoyama Cohort Study was launched in 2010, and 742 persons participated in the baseline study. Among these participants, 596 persons who attended the 2-year follow-up examinations in 2012 were evaluated, and the presence of early and late AMD was determined via grading of their fundus photographs. Based on the cohorts' data, logistic regression analyses were performed to identify the risk factors for AMD. The possible risk factors that we examined were age, sex, medical history of systemic disorders, smoking, inflammatory markers at baseline, and the complement factor H (CFH) I62V and age-related maculopathy susceptibility 2 (ARMS2) A69S variants. RESULTS: We assessed 480 participants (40.0% women) who had gradable fundus photographs. The prevalence of early AMD was 37.9% and the prevalence of late AMD was 0.6%. Mantel-Haenszel analysis revealed that the CFH I62V and ARMS2 A69S variants were significantly associated with the prevalence of AMD (P = 0.029 and 0.025, respectively). CONCLUSIONS: The CFH I62V and ARMS2 A69S variants were significantly associated with the prevalence of AMD. (www.umin.ac.jp/ctr number, UMIN000014520.).

Excessive retinol intake exacerbates choroidal neovascularization through upregulated vascular endothelial growth factor in retinal pigment epithelium in mice.

As a part of the visual cycle, all-trans-retinol (all-trans-ROL), the major form of vitamin A in circulating blood, is transported to the retinal pigment epithelium (RPE). All-trans-ROL is essential for normal retina function. However, recent researches have shown that excessive retinol intake can cause increase of all-trans-retinal. This can lead to the accumulation of lipofuscin, which is important in the pathogenesis of retina degeneration disease, such as dry type age-related macular degeneration (AMD). Since there are few reports regarding the involvement of all-trans-ROL in exudative AMD, we investigated the effects of all-trans-ROL in vitro and in vivo. We evaluated vascular endothelial growth factor (VEGF) expression in ARPE-19 cells and THP-1 cells after all-trans-ROL treatment using ELISA and real-time RT-PCR. In-vitro tube formation assay was performed with HUVEC cells using the conditioned medium (CM) obtained from ARPE-19 cells treated with all-trans-ROL. Transcriptional activity of retinoic acid receptor (RAR) was evaluated using luciferase assay. In mice, VEGF expressions were investigated in the retina and RPE/choroid after three weeks of excessive oral retinol intake. Laser-induced choroidal neovascularization (CNV) models were evaluated after they were fed with various doses of retinol. VEGF mRNA expression and VEGF production were significantly increased in all-trans-ROL treated ARPE-19 cells, which were inhibited by an RAR antagonist LE540. In contrast, there were no significant changes in VEGF production in THP-1 cells. Transcriptional activity of RAR was upregulated by all-trans-ROL treatment in ARPE-19 cells. The CM, obtained from ARPE-19 cells treated with all-trans-ROL, induced more capillary-like tube formation than cells treated with control vehicles. In vivo, the high retinol diet group has increased VEGF expression in the RPE/choroid and larger lesion size was induced. Our results suggest that all-trans-ROL is a pro-angiogenic factor. Excessive retinoid intake may be a potential risk factor for exudative AMD.

Color vision abnormality as an initial presentation of the complete type of congenital stationary night blindness.

Patients with the complete form of congenital stationary night blindness (CSNB) often have reduced visual acuity, myopia, impaired night vision, and sometimes nystagmus and strabismus, however, they seldom complain of color vision abnormality. A 17-year-old male who was at technical school showed abnormalities in the color perception test for employment, and was referred to our hospital for a detailed examination. He had no family history of color vision deficiency and no other symptoms. During the initial examination, his best-corrected visual acuity was 1.2 in both eyes. His fundus showed no abnormalities except for somewhat yellowish reflex in the fovea of both eyes. Electroretinogram (ERG) showed a good response in cone ERG and 30 Hz flicker ERG, however, the bright flash, mixed rod and cone ERG showed a negative type with a reduced b-wave (positive deflection). There was no response in the rod ERG, either. From the findings of the typical ERG, the patient was diagnosed with complete congenital stationary night blindness. This case underscores the importance of ERG in order to diagnose the cause of a color vision anomaly.

[Novel gene transfer using micellar nanovectors inhibits choroidal neovascularization].

The treatment of age-related macular degeneration (AMD) caused by choroidal neovascularization (CNV) is difficult. More effective therapy for regulating CNV is needed. We demonstrated that intravenous nonviral vectors based on the complex of plasmid DNA with synthetic cationic polymers accumulate in choroidal neovascularization (CNV) with high efficiency through an enhanced the permeability and retention (EPR) effect. This review shows the results of in vivo angiogenic control by intravenous injection of a polyplex micelle-encapsulating plasmid vector using a mice CNV model. Polyion complex (PIC) micelles consisting of plasmid DNA and poly (ethylene glycol)-b-poly (N-[N-(2-aminoethyl)-2-aminoethyl] aspartamidef block copolymers [PEG-b-PAsp (DET)] were used. These show minimal cytotoxicity and high transfection efficiency both in vitro and in vivo, and have been utilized for gene therapy against a mouse corneal neovascularization model by local administration of plasmid-encoding soluble vascular endothelial growth factor receptor 1 (soluble Fms-like tyrosine kinase-1: sFlt-1). Transfection of plasmid-expressing sFlt-1 with PEG-C6-P[Asp (DET)] polyplex micelles by intravenous injection into mice CNV models showed significant inhibition of developing CNV. We found that nonviral gene therapy has significant potential for regulation of CNV using plasmids with PEG-C6-P [Asp (DET)] polyplex micelles.

Alu-derived cis-element regulates tumorigenesis-dependent gastric expression of GASDERMIN B (GSDMB).

GASDERMIN B (GSDMB) belongs to the novel gene family GASDERMIN (GSDM). All GSDM family members are located in amplicons, genomic regions often amplified during cancer development. Given that GSDMB is highly expressed in cancerous cells and the locus resides in an amplicon, GSDMB may be involved in cancer development and/or progression. However, only limited information is available on GSDMB expression in tissues, normal and cancerous, from cancer patients. Furthermore, the molecular mechanisms that regulate GSDMB expression in gastric tissues are poorly understood. We investigated the spatiotemporal expression patterns of GSDMB in gastric cancer patients and the 5' regulatory sequences upstream of GSDMB. GSDMB was not expressed in the majority of normal gastric-tissue samples, and the expression level was very low in the few normal samples with GSDMB expression. Most pre-cancer samples showed moderate GSDMB expression, and most cancerous samples showed augmented GSDMB expression. Analysis of genome sequences revealed that an Alu element resides in the 5' region upstream of GSDMB. Reporter assays using intact, deleted, and mutated Alu elements clearly showed that this Alu element positively regulates GSDMB expression and that a putative IKZF binding motif in this element is crucial to upregulate GSDMB expression.

A new Gsdma3 mutation affecting anagen phase of first hair cycle.

Recombination-induced mutation 3 (Rim3) is a spontaneous mouse mutation that exhibits dominant phenotype of hyperkeratosis and hair loss. Fine linkage analysis of Rim3 and sequencing revealed a novel single point mutation, G1124A leading to Ala348Thr, in Gsdma3 in chromosome 11. Transgenesis with BAC DNA harboring the Rim3-type Gsdma3 recaptured the Rim3 phenotype, providing direct evidence that Gsdma3 is the causative gene of Rim3. We examined the spatial expression of Gsdma3 and characterized the Rim3 phenotype in detail. Gsdma3 is expressed in differentiated epidermal cells in the skin, but not in the proliferating epidermal cells. Histological analysis of Rim3 mutant showed hyperplasia of the epidermal cells in the upper hair follicles and abnormal anagen phase at the first hair cycle. Furthermore, immunohistochemical analysis revealed hyperproliferation and misdifferentiation of the upper follicular epidermis in Rim3 mutant. These results suggest that Gsdma3 is involved in the proliferation and differentiation of epidermal stem cells.

Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner.

Gasdermin (Gsdm) was originally identified as a candidate causative gene for several mouse skin mutants. Several Gsdm-related genes sharing a protein domain with DFNA5, the causative gene of human nonsyndromic hearing loss, have been found in the mouse and human genomes, and this group is referred to as the DFNA5-Gasdermin domain family. However, our current comparative genomic analysis identified several novel motifs distinct from the previously reported domain in the Gsdm-related genes. We also identified three new Gsdm genes clustered on mouse chromosome 15. We named these genes collectively the Gsdm family. Extensive expression analysis revealed exclusive expression of Gsdm family genes in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner. Further database searching revealed the presence of other related genes with a similar N-terminal motif. These results suggest that the Gsdm family and related genes have evolved divergent epithelial expression profiles.

Chordoid meningioma--case report.

A 69-year-old female presented with a de novo lesion detected incidentally. Computed tomography demonstrated an isodense mass in the left parietal convexity with peritumoral edema, with homogeneous enhancement by contrast medium. Magnetic resonance imaging showed the left parietal convexity tumor as isointense on T(1)-weighted imaging and homogeneously hyperintense on T(2)-weighted imaging, with homogeneous enhancement and dural tail sign after intravenous administration of gadolinium-diethylenetriaminepenta-acetic acid. The tumor was totally removed. The histological diagnosis was chordoid meningioma. Combined immunohistochemical staining was helpful to differentiate chordoid meningioma from other chordoid neoplasm.

Adequate cerebral perfusion pressure during rewarming to prevent ischemic deterioration after therapeutic hypothermia.

Ischemic deterioration during rewarming is one of the most notable clinical complications after successful therapeutic cerebral hypothermia, but the mechanism is not completely understood. Hypothermia may cause vasoconstriction and relative ischemia, especially with insufficient cerebral perfusion pressure (CPP). Various parameters were evaluated to determine the critical CPP threshold to avoid ischemia during rewarming. Cat experimental head injury was induced by inflating an epidural rubber balloon, and intracranial pressure was maintained at 30 mmHg. During rewarming after cerebral hypothermia, CPP was maintained at >120 mmHg (n = 16), 90 mmHg (n = 11), 60 mmHg (n = 11), and 40 mmHg (n=4) by controlling the blood pressure. Cerebral blood flow, cerebral metabolic rate for oxygen, arteriovenous difference of oxygen (AVDO2), cerebral venous oxygen saturation (ScvO2), and extracellular glutamate concentrations were monitored by glutamate oxidase electrode. After rewarming, the cerebral metabolic parameters were almost restored to the pre-injury level in animals with CPP of more than 90mmHg. However, in the animals with CPP= 60 mmHg, all parameters significantly deteriorated and indicated misery perfusion; ScvO2 was low (29.5+/-1.1%), AVDO2 was significantly high (9.9+/-0.8 ml 100 g(-1) min(-1)) (one-way analysis of variance, p<0.05), and electron microscopic features showed subcellular ischemic change. Extracellular glutamate significantly increased during the rewarming period only in the CPP= 40 mmHg group. CPP less than 60 mmHg during rewarming causes secondary ischemic insult, which might indicate continuation of cerebral vasoconstriction in hypothermia. CPP higher than 90 mmHg is required to avoid the potential risk of relative ischemia after hypothermia.