INCIDENCE AND RISK FACTORS OF INTRAOCULAR INFLAMMATION AFTER BROLUCIZUMAB TREATMENT IN JAPAN: A Multicenter Age-Related Macular Degeneration Study.

PURPOSE: To investigate the incidence of intraocular inflammation (IOI) and its risk factors following intravitreal injections of brolucizumab for neovascular age-related macular degeneration in Japan. METHODS: A total of 1,351 Japanese consecutive patients with neovascular age-related macular degeneration who were treated with brolucizumab from May 2020 to May 2022 at 14 institutions were examined. The variables analyzed were the number of brolucizumab injections, time to onset of IOI, and risk factors. RESULTS: Intraocular inflammation developed in 152 eyes (11.3%). Retinal vasculitis and/or retinal occlusion occurred in 53 eyes (3.9%). Ninety-four patients received bilaterally, bilateral IOI occurred in five patients (5.3%). Sixteen eyes (1.2%) had irreversible visual acuity loss and nine eyes (0.67%) had visual loss of three lines or more due to retinal vasculitis and/or retinal occlusion. The cumulative IOI incidence was 4.5%, 10.3%, and 12.2% at 30, 180, and 365 days (1-year), respectively. History of IOI (including retinal vasculitis) and/or retinal occlusion (odds ratio [OR], 5.41; P = 0.0075) and female sex (OR, 1.99; P = 0.0004) were significantly associated with IOI onset. CONCLUSION: The 1-year cumulative incidence of IOI in Japanese neovascular age-related macular degeneration patients treated with brolucizumab was 12.2%. History of IOI (including retinal vasculitis) and/or retinal occlusion and female sex were significant risk factors.

Hyperreflective material in patients with non-neovascular pachychoroid disease.

BACKGROUND: This study aimed to report eleven cases of non-neovascular pachychoroid disease with hyperreflective material (HRM) that occurred in Japanese patients. METHODS: A retrospective review of data from eleven patients who had non-neovascular retinal pigment epithelium (RPE) protrusion with HRM in the neurosensory retina between March 2017 and June 2022 was conducted. Clinical examination, color fundus photography, fluorescein angiography, spectral-domain optical coherence tomography (SD-OCT), and OCT angiography data were analyzed. Main outcome measures were patient characteristics, changes in SD-OCT findings, and symptom outcomes. RESULTS: All cases had RPE protrusion and HRM with dilated choroidal veins, which were characteristic of pachychoroid disease. However, none of the cases had macular neovascularization (MNV). In 9 eyes (81.8%), HRM improved spontaneously without intervention and resulted in alterations in RPE, referred to as pachychoroid pigment epitheliopathy (PPE) or focal choroidal excavation (FCE). In these cases, symptoms such as metamorphopsia and distortion improved without treatment. In the remaining two cases (18.2%), HRM still persisted during the follow-up period. CONCLUSION: There are some cases of non-neovascular pachychoroid disorder with HRM, which might be a new entity of pachychoroid spectrum disease or an early stage of PPE or FCE. These cases should not be misdiagnosed as MNV, and careful observation is necessary.

Mib1 contributes to persistent directional cell migration by regulating the Ctnnd1-Rac1 pathway.

Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiquitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1ta52b mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knockdown of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1ta52b mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway.

Downregulation of renal type IIa sodium-dependent phosphate cotransporter during lipopolysaccharide-induced acute inflammation.

The type IIa sodium-dependent phosphate cotransporter (Npt2a) plays a critical role in reabsorption of inorganic phosphate (Pi) by renal proximal tubular cells. Pi abnormalities during early stages of sepsis have been reported, but the mechanisms regulating Pi homeostasis during acute inflammation are poorly understood. We examined the regulation of Pi metabolism and renal Npt2a expression during lipopolysaccharide (LPS)-induced inflammation in mice. Dose-response and time-course studies with LPS showed significant increases of plasma Pi and intact parathyroid hormone (iPTH) levels and renal Pi excretion, while renal calcium excretion was significantly decreased. There was no difference in plasma 1,25-dihydroxyvitamin D levels, but the induction of plasma intact fibroblast growth factor 23 levels peaked 3 h after LPS treatment. Western blotting, immunostaining, and quantitative real-time PCR showed that LPS administration significantly decreased Npt2a protein expression in the brush border membrane (BBM) 3 h after injection, but there was no change in renal Npt2a mRNA levels. Moreover, tumor necrosis factor-α injection also increased plasma iPTH and decreased renal BBM Npt2a expression. Importantly, we revealed that parathyroidectomized rats had impaired renal Pi excretion and BBM Npt2a expression in response to LPS. These results suggest that the downregulation of Npt2a expression in renal BBM through induction of plasma iPTH levels alter Pi homeostasis during LPS-induced acute inflammation.

Cytoplasmic streaming induced by intracytoplasmic spindle translocation contributes to developmental competence through mitochondrial distribution in mouse oocytes.

OBJECTIVE: To evaluate the developmental competency of mouse metaphase II oocytes and the pattern of mitochondrial positioning through cytoplasmic streaming in mouse metaphase II oocytes. DESIGN: We observed cytoplasmic streaming as movement indicated by fluorescently stained mitochondria using a newly developed method in which the spindle is translocated to the opposite site of the oocyte. This method is termed as intracytoplasmic spindle translocation (ICST). SETTING: University research laboratory. ANIMALS: Female B6D2F1 mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fresh oocytes, postovulatory-aged oocytes, and oocytes treated with cytochalasin B were classified based on the presence of cytoplasmic streaming induced by ICST. The pattern of redistributed mitochondria and developmental competence caused by parthenogenetic activation were evaluated in oocytes with or without cytoplasmic streaming. RESULT(S): Induced cytoplasmic streaming occurred in 84% of the fresh oocytes but not in the postovulatory-aged oocytes and the oocytes treated with cytochalasin B. Abnormal mitochondrial aggregation was observed in oocytes in which cytoplasmic streaming was not induced. Furthermore, the developmental competence was significantly lower in oocytes without cytoplasmic streaming. CONCLUSION(S): Cytoplasmic streaming induced by ICST contributes to developmental competence through the redistribution of mitochondria and may be a valuable criterion for predicting early developmental competence in mouse oocytes.

Short-term outcomes of switching to brolucizumab in japanese patients with neovascular age-related macular degeneration.

PURPOSE: To evaluate the outcomes of a 6-month follow-up after switching to brolucizumab from aflibercept to treat neovascular age-related macular degeneration (AMD) in Japanese patients. STUDY DESIGN: Retrospective observational study. METHODS: We studied 45 consecutive eyes of 42 patients diagnosed with neovascular AMD, who were switched to intravitreal brolucizumab injection (IVBr) after receiving intravitreal aflibercept injection (IVA) using a treat-and-extend (TAE) regimen. Patients who had brolucizumab-associated intraocular inflammation (IOI) were excluded from the study. The mean changes in the logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA), central foveal thickness (CFT), central choroidal thickness (CCT), and treatment intervals were evaluated at 6 months after the switch to IVBr. RESULTS: One eye of 1 patient was excluded because of IOI after the switch; 44 eyes of 41 patients were enrolled in this study. The mean logMAR BCVA was maintained throughout the follow-up period when compared with the baseline value (P > .05 at 6 months). However, the mean CFT and CCT at 6 months had decreased significantly (P < .05 and P < .001, respectively). The mean treatment interval was extended from 5.75 to 8.12 weeks. CONCLUSION: Switching to brolucizumab from aflibercept using a TAE regimen might be effective for maintaining functional outcomes and extending intervals in Japanese patients with AMD.

One-year outcomes of intravitreal brolucizumab injections in patients with polypoidal choroidal vasculopathy.

To evaluate the 1-year visual outcomes and anatomic responses of Japanese patients who received intravitreal brolucizumab (IVBr) injections for polypoidal choroidal vasculopathy (PCV). This was a retrospective study of 17 treatment-naïve eyes with PCV that were treated with IVBr. We evaluated the best-corrected visual acuity (BCVA), central macular thickness (CMT), central choroidal thickness (CCT) and number of injections for 1 year. The eradication of polypoidal lesions was also evaluated using by indocyanine green angiography during the 1-year follow-up. Non-infectious intraocular inflammation developed in two (11.8%) eyes; 15 eyes were assessed at the 1-year follow-up examination. The mean BCVA improved significantly from 0.28 at baseline to 0.13 (P < 0.05) at 1 year. The CMT and CCT decreased significantly after 1 year. The mean number of injections was 6.4 ± 0.13. The rate of complete resolution of polypoidal lesions at 1 year was 93.3%. A dry macula was achieved in 13 eyes (86.6%) after the loading phase and in 11 eyes (73.3%) at 1 year. The IVBr injections appeared to be effective for improving both functional and anatomic outcomes in Japanese patients with PCV, with a high regression rate of polypoidal lesions.

Regulation of renal sodium-dependent phosphate co-transporter genes (Npt2a and Npt2c) by all-trans-retinoic acid and its receptors.

The type II sodium-dependent phosphate co-transporters Npt2a and Npt2c play critical roles in the reabsorption of Pi by renal proximal tubular cells. The vitamin A metabolite ATRA (all-trans-retinoic acid) is important for development, cell proliferation and differentiation, and bone formation. It has been reported that ATRA increases the rate of Pi transport in renal proximal tubular cells. However, the molecular mechanism is still unknown. In the present study, we observed the effects of a VAD (vitamin A-deficient) diet on Pi homoeostasis and the expression of Npt2a and Npt2c genes in rat kidney. There was no change in the plasma levels of Pi, but VAD rats significantly increased renal Pi excretion. Renal brush-border membrane Pi uptake activity and renal Npt2a and Npt2c expressions were significantly decreased in VAD rats. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of human Npt2a and Npt2c genes was increased markedly by ATRA and a RAR (retinoic acid receptor)-specific analogue TTNPB {4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid} in renal proximal tubular cells overexpressing RARs and RXRs (retinoid X receptors). Furthermore, we identified RAREs (retinoic acid-response elements) in both gene promoters. Interestingly, the half-site sequences (5'-GGTTCA-3': -563 to -558) of 2c-RARE1 overlapped the vitamin D-responsive element in the human Npt2c gene and were functionally important motifs for transcriptional regulation of human Npt2c by ATRA and 1,25(OH)2D3 (1alpha,25-dihydroxyvitamin D3), in both independent or additive actions. In summary, we conclude that VAD induces hyperphosphaturia through the down-regulation of Npt2a and Npt2c gene expression in the kidney.

Incidence of elevated intraocular pressure after intravitreal injection in Japanese patients with age-related macular degeneration.

The purpose of this study was to report the incidence of elevated intraocular pressure (IOP) after intravitreal injection (IVI) of anti-vascular endothelial growth factor (VEGF) in Japanese patients with age-related macular degeneration (AMD). A retrospective study of chart review of patients who underwent ≥ 10 intravitreal anti-VEGF injections between April 2009 and December 2019 was conducted. Elevated IOP was defined as IOP ≥ 25 mmHg at one visit. Cases with elevated IOP resulting from IVI were identified. Furthermore, the association between elevated IOP and some parameters, as the risk factors that influence elevated IOP, was investigated. A total of 402 eyes of 370 patients were included in this study. Twenty-eight eyes of 26 patients (7.0%) were identified as cases with elevated IOP after IVI. The mean time of elevation after baseline was 50.6 ± 26.5 months. History of glaucoma (p = 0.021; odds ratio, 5.85), treatment modality (p = 0.019; odds ratio, 6.32), and total number of injections (p = 0.003; odds ratio, 1.03) were significantly associated with elevated IOP. A late complication of elevated IOP is associated with IVI in patients with AMD. Particularly, history of glaucoma and treat and extend regimen with frequent injections were found to be risk factors of elevated IOP.

Prenatal diagnosis of Fraser syndrome caused by novel variants of FREM2.

Fraser syndrome (FS) involves multiple malformations and has a 25% recurrence risk among siblings. However, these malformations are difficult to detect prenatally, hampering prenatal diagnosis. Here, we describe a fetus with FS diagnosed using ultrasonography. Ultrasonography revealed congenital high airway obstruction syndrome and renal agenesis. Syndactyly of both hands and cryptophthalmos were noted postnatally, and the diagnosis was confirmed by genetic analysis, which showed novel compound heterozygous variants of FREM2.

Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.

Actin cytoskeleton reorganization is a fundamental process for actin-based cellular functions such as cytokinesis, phagocytosis, and chemotaxis. Regulating actin cytoskeleton reorganization is therefore an attractive approach to control endothelial and inflammatory cells function and to treat cardiovascular diseases. Here, we identified glia maturation factor-gamma (GMFG) as a novel factor in actin cytoskeleton reorganization and is expressed preferentially in microvascular endothelial and inflammatory cells. During mouse embryogenesis, GMFG was expressed predominantly in blood islands of the yolk sac, where endothelial and hematopoietic cells develop simultaneously. In endothelial cells, GMFG was colocalized with F-actin in membrane ruffles and was associated with F-actin assessed by actin co-sedimentation assay. Interestingly, GMFG was phosphorylated at N-terminal serine, and its phosphorylation was enhanced by coexpression of dominant active Rac1 and Cdc42. Furthermore, a pseudophosphorylated form of GMFG (GMFG-S2E) demonstrated higher association with F-actin. Stable expression of GMFG-S2E remarkably enhanced stimulus-responsive lamellipodia and subsequent membrane ruffle formation in HeLa cells presumably through its interaction with Arp2/3 complex. Expression of GMFG enhanced actin-based cellular functions such as migration and tube-formation in endothelial cells. Moreover, we found that GMFG expression was significantly increased in a cardiac ischemia/reperfusion model where inflammation and angiogenesis take place actively. Taken together, our findings define a novel pathway in the regulation of actin-based cellular functions. Regulating GMFG function may provide a novel approach to modulate the pathophysiology of cardiovascular diseases.

Dietary phosphate supplementation delays the onset of iron deficiency anemia and affects iron status in rats.

Inorganic phosphate (Pi) plays critical roles in bone metabolism and is an essential component of 2,3-diphosphoglycerate (2,3-DPG). It has been reported that animals fed a low-iron diet modulate Pi metabolism, whereas the effect of dietary Pi on iron metabolism, particularly in iron deficiency anemia (IDA), is not fully understood. In this study, we hypothesized the presence of a link between Pi and iron metabolism and tested the hypothesis by investigating the effects of dietary Pi on iron status and IDA. Wistar rats aged 4 weeks were randomly assigned to 1 of 4 experimental dietary groups: normal iron content (Con Fe)+0.5% Pi, low-iron (Low Fe)+0.5% Pi, Con Fe+1.5% Pi, and Low Fe+1.5% Pi. Rats fed the 1.5% Pi diet for 14 days, but not for 28 days, maintained their anemia state and plasma erythropoietin concentrations within the reference range, even under conditions of low iron. In addition, plasma concentrations of 2,3-DPG were significantly increased by the 1.5% Pi diets and were positively correlated with plasma Pi concentration (r=0.779; P<.001). Dietary Pi regulated the messenger RNA expression of iron-regulated genes, including divalent metal transporter 1, duodenal cytochrome B, and hepcidin. Furthermore, iron concentration in liver tissues was increased by the 1.5% Pi in Con Fe diet. These results suggest that dietary Pi supplementation delays the onset of IDA and increases plasma 2,3-DPG concentration, followed by modulation of the expression of iron-regulated genes.

Dietary phosphate restriction induces hepatic lipid accumulation through dysregulation of cholesterol metabolism in mice.

Excessive inorganic phosphate (Pi) intake and hyperphosphatemia have both been speculated to be risk factors for cardiovascular disease and hypercholesterolemia, and dysregulation of cholesterol metabolism can lead to atherosclerosis. However, the relationship between Pi and cholesterol metabolism has not been investigated in detail. Our recent study showed that triiodothyronine can induce both hyperphosphatemia and hypocholesterolemia in mice. We therefore hypothesized a possible linkage between Pi and cholesterol metabolism. In this study, we investigated the effects of dietary Pi intake on cholesterol metabolism in mice. Mice were divided into 4 groups, which were fed diets containing 1.2% or 0.1% Pi and with or without 2% cholesterol (Pi-sufficient, Pi-restricted, Pi-sufficient + Chol, and Pi-restricted + Chol), for 12 days. Inorganic phosphate-restricted mice exhibited significantly higher liver weights than did Pi-sufficient mice. Interestingly, dietary Pi restriction significantly increased high-cholesterol diet-induced hepatic lipid accumulation. Real-time polymerase chain reaction analysis revealed that dietary Pi restriction decreased expression of hepatic genes involved in cholesterol metabolism and fatty acid biosynthesis. In addition, hepatic messenger RNA levels of several transcription factors including peroxisome proliferator-activated receptors and liver X receptor were markedly decreased by Pi restriction. Furthermore, plasma lipid and lipoprotein profile analysis showed that dietary Pi restriction reduced susceptibility to high-cholesterol diet-induced hyperlipidemia. Importantly, we found that there was a significant negative correlation between plasma levels of Pi and total cholesterol. These results suggest that dietary Pi plays an important role in the development of fatty liver disease and hyperlipidemia induced by a high-cholesterol diet through regulation of lipid metabolism-related gene expression in the liver.

Hypercholesterolemia and effects of high cholesterol diet in type IIa sodium-dependent phosphate co-transporter (Npt2a) deficient mice.

The type IIa sodium-dependent phosphate co-transporter (Npt2a) is important to maintain renal inorganic phosphate (Pi) homeostasis and the plasma Pi levels. It has reported that disorder of Pi metabolism in kidney can be risk factors for cardiovascular disease as well as hypercholesterolemia. However, the relationship between Pi and cholesterol metabolism has not been clarified. The current study investigated the effects of Npt2a gene ablation that is known as hypophosphatemia model on cholesterol metabolism in mice. Npt2a deficient (Npt2a(-/-)) mice and wild type mice were fed diets with or without 2% cholesterol for 12 days. Plasma lipid and lipoprotein profile analysis revealed that plasma lipid levels (total, LDL and HDL cholesterol) were significantly higher in Npt2a(-/-) mice than wild type (WT) mice. Interestingly, high cholesterol diet markedly increased plasma levels of total, LDL and HDL cholesterol in WT mice, but not Npt2a(-/-) mice. On the other hand, there were no differences in body and liver weight, intake and hepatic lipid accumulation between WT and Npt2a(-/-) mice. These results suggest that ablation of Npt2a gene induces hypercholesterolemia and affects the ability to respond normally to dietary cholesterol.

Thyroid hormones decrease plasma 1α,25-dihydroxyvitamin D levels through transcriptional repression of the renal 25-hydroxyvitamin D3 1α-hydroxylase gene (CYP27B1).

The primary determinant of circulating 1α,25-dihydroxyvitamin D (1,25[OH](2)D) levels is the activity of 25-hydroxyvitamin D-1α-hydroxylase (cytochrome P450 27B1 [CYP27B1]) in the kidney. Hyperthyroid patients have been reported to have low levels of plasma 1,25(OH)(2)D. However, the detailed mechanism of thyroid hormone action on vitamin D metabolism is still poorly understood. The present study determined whether renal CYP27B1 gene expression was negatively regulated by thyroid hormones. T(3)-induced hyperthyroid mice showed marked decreases in plasma 1,25(OH)(2)D levels and in renal expression of CYP27B1 mRNA but no changes in plasma concentrations of calcium, PTH, or fibroblast growth factor-23. In addition, we observed that T(3) administration significantly decreased plasma 1,25(OH)(2)D and renal CYP27B1 mRNA levels that were increased by low-calcium or low-phosphorus diets and induced hypocalcemia in mice fed a low-calcium diet. Promoter analysis revealed that T(3) decreases the basal transcriptional activity of the CYP27B1 gene through thyroid hormone receptors (TRα and TRß1) and the retinoid X receptor α (RXRα) in renal proximal tubular cells. Interestingly, we identified an everted repeat negative thyroid hormone response element (1α-nTRE) overlapping the sterol regulatory element (1α-SRE) and the TATA-box -50 to -20 base pairs from the human CYP27B1 gene transcription start site. Finally, we established that CYP27B1 gene transcription is positively regulated by SRE-binding proteins and that a T(3)-bound TRß1/RXRα heterodimer inhibits SRE-binding protein-1c-induced transcriptional activity through the 1α-nTRE. These results suggest that transcriptional repression of the CYP27B1 gene by T(3)-bound TRs/RXRα, acting through the 1α-nTRE, results in decreased renal CYP27B1 expression and plasma 1,25(OH)(2)D levels.

Up-regulation of stanniocalcin 1 expression by 1,25-dihydroxy vitamin D(3) and parathyroid hormone in renal proximal tubular cells.

Stanniocalcin 1 and stanniocalcin 2 are two glycoprotein hormones, which act as calcium phosphate-regulating factor on intestine and kidney. We have previously reported that stanniocalcin 2 expression is positively and negatively controlled by 1,25(OH)(2)D(3) and parathyroid hormone in renal proximal tubular cells. However, it has been unclear whether they regulate the stanniocalcin 1 gene expression. In this study, we identified the opossum stanniocalcin 1 cDNA sequence. The opossum stanniocalcin 1 amino acid sequence had 83% homology with human stanniocalcin 1, and has a conserved putative N-linked glycosylation site. Real-time PCR analysis using opossum kidney proximal tubular (OK-P) cells revealed that the mRNA levels of stanniocalcin 1 gene is up-regulated by both 1,25(OH)(2)D(3) and parathyroid hormone in dose-dependent and time-dependent manners. We also demonstrated that the stanniocalcin 1 expression was increased in parathyroid hormone injected rat kidney. Furthermore, the mRNA expression of stanniocalcin 1 and stanniocalcin 2 were oppositely regulated by phorbol 12,13-myristic acetate, a specific PKC activator. Interestingly, the up-regulation of stanniocalcin 1 gene by 1,25(OH)(2)D(3) and phorbol 12,13-myristic acetate were not prevented in the presence of actinomycin D, an RNA synthesis inhibitor. These results suggest that the stanniocalcin 1 gene expression is up-regulated by 1,25(OH)(2)D(3) and parathyroid hormone through mRNA stabilization in renal proximal tubular cells.

Self-recognition of N-linked glycans with multivalent GlcNAc, determined as ceramide mimetic conjugate.

Aminoceramide mimetic was synthesized and conjugated to N-linked oligosaccharides having multivalent GlcNAc by reductive amination. Ceramide mimetic conjugates with "complex-type" glycan having five or six GlcNAc termini (termed Os Fr. B-Cer) were purified, analyzed by thin-layer chromatography (TLC), and finally characterized by MS/MS analysis through liquid chromatography/mass spectrometry. Binding of Os Fr. B-Cer placed on solid phase polystyrene surface with [3H]cholesterol-labeled liposomes containing Os Fr. B-Cer, or containing various glycosphingolipids (GSLs) was determined. The binding of Os Fr. B-Cer liposomes to Os Fr. B-Cer coated plate was significantly higher than binding of GM3 liposomes. Other GSL liposomes showed no binding. Thus, self-recognition of Os Fr. B-Cer was clearly demonstrated using ceramide mimetic conjugates.

Molecular isolation and characterization of novel four subisoforms of ECE-2.

Endothelin-converting enzymes (ECEs) are the key enzymes in the endothelin (ET) biosynthesis that catalyze the conversion of big ET, the biologically inactive precursor of mature ET. Two enzymes, termed ECE-1 and ECE-2, have been molecularly identified. Here, we report novel four subisoforms of ECE-2 that differ in their N-terminal cytoplasmic tails, termed ECE-2a-1, ECE-2a-2, ECE-2b-1, and ECE-2b-2. RT-PCR analysis of these subisoforms in bovine tissues demonstrated that their tissue distribution was strikingly different. ECE-2a-1 and ECE-2a-2 are expressed in a variety of tissues including liver, kidney, adrenal gland, testis, and endothelial cells, while ECE-2b-1 and ECE-2b-2 are expressed abundantly in brain and adrenal gland. Furthermore, ECE-2a-1 and ECE-2b-2 were revealed to be predominant forms as compared to ECE-2a-2 and ECE-2b-1, respectively. Immunohistochemical analyses of CHO cells, stably expressing ECE-2a-1 or ECE-2b-2, revealed that both ECE-2a-1 and ECE-2b-2 were localized in intracellular compartments but not on the cell surface. Detailed analysis of ECE-2 subisoforms will provide crucial information to clarify the physiological function of ECE-2.