Does administration of hydroxychloroquine/amiodarone affect the efficacy of enzyme replacement therapy for Fabry mice?

As a standard therapy for Fabry disease, enzyme replacement therapy (ERT) with recombinant human α-galactosidase A (α-Gal) has been successfully used, and the instructions for this drug state that "it should not be co-administrated with cationic amphiphilic drugs such as hydroxychloroquine (HCQ) and amiodarone (AMI), since these drugs have the potential to inhibit intracellular α-Gal activity". However, there would be cases in which HCQ or AMI is required for patients with Fabry disease, considering their medical efficacy and application. Thus, we examined the impact of HCQ/AMI on recombinant human α-Gal by in vitro, cellular, and animal experiments. The results revealed that HCQ/AMI affected the enzyme activity of α-Gal incorporated into cultured fibroblasts from a Fabry mouse when the cells were cultured in medium containing these drugs and the enzyme, although their direct inhibitory effect on the enzyme is not strong. These lysosomotropic drugs may be trapped and concentrated in lysosomes, followed by inhibition of α-Gal. On the other hand, no reduction of α-Gal activity incorporated into the organs and tissues, or acceleration of glycoshingolipid accumulation was observed in Fabry mice co-administered with HCQ/AMI and the enzyme, compared with in the case of usual ERT. As HCQ/AMI administered are catabolized in the liver, these drugs possibly do not affect ERT for Fabry mice, different from in the case of cultured cells in an environment isolated from the surroundings.

Effects of switching from agalsidase-α to agalsidase-ß on biomarkers, renal and cardiac parameters, and disease severity in fabry disease forming neutralizing antidrug antibodies: a case report.

Fabry disease is an X-linked hereditary disorder caused by deficient α-galactosidase A (GLA) activity. Patients with Fabry disease are often treated with enzyme replacement therapy (ERT). However, ERT often induces the formation of neutralizing antidrug antibodies (ADAs), which may impair the therapeutic efficacy. Here, we report the case of a 32-year-old man with Fabry disease and resultant neutralizing ADAs who was treated by switching from agalsidase-α to agalsidase-ß. We monitored biomarkers, such as plasma globotriaosylsphingosine (lyso-Gb3), urinary globotriaosylceramide (Gb3), urinary mulberry bodies, renal and cardiac parameters, and disease severity during the treatment period. Although plasma lyso-Gb3 and urinary Gb3 levels quickly decreased within two months after the initiation of ERT with agalsidase-α, they gradually increased thereafter. The urinary mulberry bodies continued to appear. Both the ADA titer and serum mediated GLA inhibition rates started to increase after two months. Moreover, 3.5 years after ERT, the vacuolated podocyte area in the renal biopsy decreased slightly from 23.1 to 18.9%. However, plasma lyso-Gb3 levels increased, and urinary Gb3, mulberry body levels, and ADA titers remained high. Therefore, we switched to agalsidase-ß which reduced, but did not normalize, plasma lyso-Gb3 levels and stabilized renal and cardiac parameters. Disease severity was attenuated. However, urinary Gb3 and mulberry body levels did not decrease noticeably in the presence of high ADA titers. The kidneys take up a small amount of the administered recombinant enzyme, and the clearance of Gb3 that has accumulated in the kidney may be limited despite the switching from agalsidase-α to agalsidase-ß.

Profiles of Globotriaosylsphingosine Analogs and Globotriaosylceramide Isoforms Accumulated in Body Fluids from Various Phenotypic Fabry Patients.

Objectives Fabry disease is characterized by the systemic accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), which are widely used as biomarkers of the disease. However, few reports have described the relationship of Lyso-Gb3 analogs and Gb3 isoforms with the disease. The present study determined the profiles of Lyso-Gb3 analogs and Gb3 isoforms accumulated in body fluids from various phenotypic Fabry patients to elucidate the basis of the disease. Methods Plasma Lyso-Gb3 and related analogs were measured in 15 classic Fabry men, 6 later-onset Fabry men, 11 Fabry women, and 36 controls, while urinary Gb3 isoforms were measured in 5 classic Fabry men, 5 later-onset Fabry men, 17 Fabry women, and 11 controls, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, these values were monitored for a classic Fabry man, in whom neutralizing anti-drug antibodies had developed following enzyme replacement therapy (ERT). Results The levels of plasma Lyso-Gb3 analogs/urinary Gb3 isoforms were higher in Fabry patients than in controls, especially in classic Fabry men. However, minor differences in the ratio of each Lyso-Gb3 analog and Gb3 isoform with respect to the total Lyso-Gb3 analogs and Gb3 isoforms, respectively, were observed among individual classic Fabry men. Their time courses were well associated with the development and attenuation of anti-drug antibodies in a patient with classic Fabry disease during ERT. Conclusion Quantification of Lyso-Gb3 analogs and Gb3 isoforms provides us with more detailed information about the substrates that accumulated in the body fluids of Fabry patients than does quantification of Lyso-Gb3 and Gb3 alone, so this approach may be useful for elucidating the basis of Fabry disease.

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of α-galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified α-N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.

Monitoring of anti-drug antibodies and disease-specific biomarkers in three patients from a Japanese Fabry family treated with enzyme replacement therapy.

We monitored anti-drug antibodies and disease-specific biomarkers in three patients with a nonsense mutation from a Japanese Fabry family treated with enzyme replacement therapy (ERT). In two male patients from the family, neutralizing anti-drug antibodies were induced at an early stage of ERT, the antibody titer peak being found at an earlier stage of ERT in the patient treated with 1.0 mg/kg agalsidase beta than in that treated with 0.2 mg/kg agalsidase alfa. Then, the antibody titers decreased with continuation of ERT. The formation of neutralizing anti-drug antibodies adversely affected the plasma globotriaosylsphingosine (Lyso-Gb3) level and urinary globotriaosylceramide (Gb3) excretion in both patients, the impact being greater in the patient treated with 0.2 mg/kg agalsidase alfa than in that treated with 1.0 mg/kg agalsidase beta. The difference might be explained by the different doses of the infused enzymes based on supersaturation of the antibodies. In a heterozygous Fabry female from the family, no sign of antibody formation was found, and both the plasma Lyso-Gb3 level and urinary Gb3 excretion, which were moderately increased at the baseline, decreased gradually. No deterioration of the manifestations or laboratory findings was observed during ERT in either of the patients. Thus, monitoring of anti-drug antibodies and biomarkers in these Fabry patients provided us with important information on their pathological condition during ERT.

Comparative urinary globotriaosylceramide analysis by thin-layer chromatography-immunostaining and liquid chromatography-tandem mass spectrometry in patients with Fabry disease.

In Fabry disease, accumulation of glycolipids, predominantly globotriaosylceramide (Gb3), affects the kidneys, and nephropathy is one of the important disorders that influence the disease severity and prognosis of patients. Urinary Gb3 has been analyzed for diagnosis and monitoring of Fabry disease. In this study, we analyzed urinary Gb3 by thin-layer chromatography (TLC)-immunostaining and liquid chromatography (LC)-tandem mass spectrometry (MS/MS). An improved qualitative method, TLC-immunostaining, revealed excessive urinary Gb3 excretion in 100 (8/8), 88 (14/16), and 74% (45/61) of the classic Fabry males, later-onset Fabry males, and Fabry females examined, respectively. This authentic method is robust, easy, economic, and hardly affected by abundant urinary sediment, and this is useful for diagnosing individual Fabry patients. LC-MS/MS can determine the level of Gb3 in urine with high sensitivity, and it revealed that the Gb3 excretion level was higher in the order of classic Fabry males, later-onset Fabry males, Fabry females, and controls, respectively, and this is expected to be a useful quantitative method not only for diagnosis but also for predicting the progression of Fabry nephropathy. As to the relation of the urinary Gb3 level and renal events, our study revealed that the urinary Gb3 level in Fabry patients experiencing renal events tended to be higher than that in ones who did not have any renal events in each phenotypic group of the disease.

Does administration of hydroxychloroquine/amiodarone accelerate accumulation of globotriaosylceramide and globotriaosylsphingosine in Fabry mice?

Drug-induced lysosomal storage disease (DILSD) caused by cationic amphiphilic drugs (CADs), which exhibits toxic manifestations and pathological findings mimicking Fabry disease (α-galactosidase A deficiency), has attracted the interests of clinicians and pathologists. Although the affected region is lysosomes in both the diseases, DILSD is characterized by intralysosomal accumulation of phospholipids and Fabry disease that of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). However, it is unknown whether administration of CADs affects the catabolism of Gb3 and Lyso-Gb3 in Fabry disease. In this study, we independently administered hydroxychloroquine/amiodarone to wild-type and Fabry mice and examined the effects of the drugs on the enzyme activity and substrates accumulated in organs and tissues. The results revealed that the administration of the drugs induced accumulation of phosphatidylcholine in both the wild-type and Fabry mice. However, reduction of α-galactosidase A activity in the organs and tissues of the wild-type mice was not found, and the storage of Gb3 and Lyso-Gb3 was not accelerated by these drugs in the Fabry mice. This suggests that hydroxychloroquine/amiodarone do not have any significant impact on the catabolism of Gb3 and Lyso-Gb3 in organs and tissues of both wild-type and Fabry mice.

Simultaneous self-monitoring comparison of a supine algorithm-equipped wrist nocturnal home blood pressure monitoring device with an upper arm device.

A nocturnal home blood pressure (BP) monitoring device that measures nighttime BP levels accurately with less sleep disturbance is needed for the 24-h management of hypertension. Here we conducted the first comparison study of simultaneous self-monitoring by both a supine position algorithm-equipped wrist nocturnal home BP monitoring device, the HEM-9601T (NightView; Omron Healthcare) with a similar upper arm device, the HEM-9700T (Omron Healthcare) in 50 hypertensive patients (mean age 68.9 ± 11.3 years). Both devices were worn on the same non-dominant arm during sleep over two nights. The patients self-measured their nighttime BP by starting nocturnal measurement mode just before going to bed. In total, 694 paired measurements were obtained during two nights (7.2 ± 1.5 measurements per night), and the mean differences (±SD) in systolic BP between the devices was 0.2 ± 10.2 mmHg (p = .563), with good agreement. In the comparison of nighttime BP indices, the difference in average SBP at 2:00, 3:00, and 4:00 AM and the average SBP of 1-h interval measurements was -0.5 ± 5.5 mmHg (p = .337), with good agreement. The HEM-9601T substantially reduced sleep disturbance compared to the upper arm-type device. The newly developed HEM-9601T (NightView) can thus accurately measure BP during sleep without reducing the wearer's sleep quality.

Anti-drug antibody formation in Japanese Fabry patients following enzyme replacement therapy.

Enzyme replacement therapy (ERT) for Fabry disease (deficiency of α-galactosidase A, α-Gal) with recombinant α-Gals (agalsidase alfa and agalsidase beta) is widely available and improves some of the clinical manifestations and biochemical findings. However, recent reports suggest that recurrent administration of recombinant enzymes often induces the formation of anti-drug antibodies, which may have a negative impact on the outcome of the therapy. We examined the formation of anti-drug antibodies using blood samples from 97 Japanese Fabry patients following ERT and tried to characterize them by means of enzyme-linked immunosorbent assay (ELISA), serum-mediated α-Gal inhibition, and immunochromatographic (IC) assay, followed by GLA gene analysis and measurement of plasma globotriaosylsphingosine (lyso-Gb3). ELISA revealed that 20/35 (57%) classic Fabry males were antibody (Immunoglobulin G, IgG) -positive (Ab+) at 6 months after the initiation of ERT, although only two of the seventeen (12%) later-onset Fabry males and none of the 45 Fabry females were. The Ab+ state was maintained at least until 24 months after the initiation of ERT in most of the cases, the exceptions being two patients who acquired immune tolerance during ERT. As many Ab+ patients have nonsense mutations, attention should be paid to the formation of anti-drug antibodies in Fabry patients harboring such gene mutations, who hardly produce α-Gal protein. Serum-mediated α-Gal inhibition was seen in most of the Ab+ patients and the antibodies affected the reduction of the plasma lyso-Gb3 level following ERT, suggesting that the antibodies inhibit the enzyme activity. There was a correlation between the results of the IC test and those of the ELISA. As the former is easy and rapid, it should be useful as a bed-side test.

Urinary afamin levels are associated with the progression of diabetic nephropathy.

AIMS: In this study, we applied quantitative proteomic analysis to identify urinary proteins associated with diabetic nephropathy (DN). METHODS: Two-dimensional image-converted analysis of liquid chromatography and mass spectrometry detected the proteins differentially excreted between normoalbuminuric and macroalbuminuric patients with type 2 diabetes mellitus (T2DM) (n = 6 each). Urinary levels of excreted proteins were measured by multiple reaction monitoring (MRM) analysis using an independent sample set (n = 77). Urinary afamin levels were measured by ELISA in T2DM and DN patients enrolled in this cohort study (n = 203). RESULTS: One-hundred-four proteins displayed significant alterations in excretion. Nine of these candidates were validated by MRM analysis. Among them, the levels of afamin, CD44 antigen, and lysosome-associated membrane glycoprotein 2, which have not previously been implicated in DN, were significantly associated with both the urinary albumin to creatinine ratio (ACR) and eGFR. We further measured afamin levels in urine collected from T2DM patients who did not yet have significant kidney disease (ACR < 300 mg/g or eGFR change rate ≤ 3.3%/year). The urinary afamin to creatinine ratio (Afa/Cre) was significantly higher in patients who progressed to a more severe DN stage or had early renal decline than in patients who did not. CONCLUSIONS: Afa/Cre was significantly increased in T2DM patients who subsequently developed DN. Afa/Cre may be useful to predict patients with T2DM at high risk of nephropathy before the development of macroalbuminuria or reduced kidney function, although further validation studies in a larger population are needed.

Fabry disease in a Japanese population-molecular and biochemical characteristics.

We had experienced 117 Japanese Fabry patients (72 males and 45 females) from 1977 to 2006, and then we generated an improved Fabry analysis system in 2007 and have found 196 ones (95 males and 101 females) since then. In this study, we summarized the data of the patients and tried to elucidate the molecular and biochemical characteristics of Japanese Fabry patients. Gene analysis revealed various GLA mutations, including missense mutations (56.5%, 48 types); nonsense mutations (15.9%, 13 types); deletions (12.6%, 13 types); splicing defects (10.1%, 6 types); insertions (1.0%, 2 types), and insertions/deletions (0.5%, 1 type), in the patients that were tested. Amino acid substitutions resulting from the missense mutations found in the classic form patients tended to be localized in the core of the GLA protein, and those in the later-onset ones in the peripheral region. The most commonly identified pathogenic mutations are c.888G > A (p.M296I), c.936 + 919G > A, c.679C > T (p.R227X), c.335G > A (p.R112H), c.334C > T (p.R112C), and c.902G > A (p.R301Q). Among them, c.888G > A (p.M296I) is unique to Japanese Fabry patients. On the other hand, c.936 + 919G > A is a variant that has been frequently detected in Taiwan Chinese Fabry patients, and c.335G > A (p.R112H) in various countries. These are found in later-onset patients, and c.679C > T (p.R227X) and c.334C > T (p.R112C) classic ones. c.902G > A (p.R301Q) is found in both classic and later-onset form patients. A possible functional polymorphism, c.196G > C (p.E66Q), was identified in 0.4% of the subjects who underwent high-risk screening. The biochemical findings including leukocyte α-galactosidase A activity, plasma globotriaosylsphingosine level and urinary globotriaosylceramide in the individual phenotypic groups well reflected the phenotypic differences in this disease. The results will be useful for understanding the basis of Fabry disease in Japan.

Proteomic analysis of serum biomarkers for prediabetes using the Long-Evans Agouti rat, a spontaneous animal model of type 2 diabetes mellitus.

AIMS/INTRODUCTION: To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus, differential serum proteomic analysis was carried out on a spontaneous animal model of type 2 diabetes mellitus without obesity, the Long-Evans Agouti (LEA) rat. MATERIALS AND METHODS: We carried out quantitative proteomic analysis using serum samples from 8- and 16-week-old LEA and control Brown Norway (BN) rats (n = 4/group). Differentially expressed proteins were validated by multiple reaction monitoring analysis using the sera collected from 8-, 16-, and 24-week-old LEA (n = 4/each group) and BN rats (n = 5/each group). Among the validated proteins, we also examined the possible relevance of the human homolog of serine protease inhibitor A3 (SERPINA3) to type 2 diabetes mellitus. RESULTS: The use of 2-D fluorescence difference gel electrophoresis analysis and the following liquid chromatography-multiple reaction monitoring analysis showed that the serum levels of five proteins were differentially changed between LEA rats and BN rats at all three time-points examined. Among the five proteins, SERPINA3N was increased significantly in the sera of LEA rats compared with age-matched BN rats. The serum level of SERPINA3 was also found to be significantly higher in type 2 diabetes mellitus patients than in healthy control participants. Furthermore, glycated hemoglobin, fasting insulin and estimated glomerular filtration rate were independently associated with the SERPINA3 levels. CONCLUSIONS: These findings suggest a possible role for SERPINA3 in the development of the early stages of type 2 diabetes mellitus, although further replication studies and functional investigations regarding their role are required.

Obesity, metabolic syndrome.

The prevalence of reflux esophagitis was low in Japan but has increased in recent years. This increase might be due to the increase in the elderly population and obesity with west- ernized diets. Obesity and the metabolic syndrome cause reflux esophagitis. Increase in central obesity induces inflammation in fat tissue. Moreover, insulin resistance induces hypertension, hypertriglyceridemia and impaired glucose tolerance, and causes the metabolic syndrome. Anti-hypertensive drugs (calcium channel blockers, etc.) and hyperglycemia might cause an esophageal movement disorder. Inflammatory state and change of esophageal move- ment might cause and worsen reflux esophagitis. The metabolic syndrome, characterized by central obesity, is a risk factor for reflux esophagitis. It is necessary to consider lifestyle improvements and medications to decrease weight and central obesity.

Multicenter investigation of lifestyle-related diseases and visceral disorders in thalidomide embryopathy at around 50 years of age.

BACKGROUND: In utero exposure to thalidomide causes a wide range of birth defects, including phocomelia, hearing loss and visceral disorders, known as thalidomide embryopathy (TE). Fifty years after the first report of TE, we conducted the first cross-sectional multicenter study to investigate the development of lifestyle-related diseases and identify risk factors for visceral disorders in subjects with TE. METHODS: Seventy-six cases with TE (31 men, 45 women) underwent medical examinations between 2011 and 2014 to determine the types of TE-related anomalies (limbs, auditory organs, or visceral organs) and lifestyle-related diseases present. Logistic multiple regression analyses, adjusted for gender and age, were conducted between TE and lifestyle-related diseases and to evaluate association between block vertebra and gallbladder aplasia. RESULTS: Fatty liver (FL), nonalcoholic FL disease and dyslipidemia were detected in 52.6%, 35.0%, and 23.7% of subjects, respectively, with higher incidences among men. Dyslipidemia was detected in 40.0% of subjects with FL and was significantly associated with FL (odds ratio = 8.86; p = 0.008). Block vertebrae were detected in 44.4% of subjects with gallbladder aplasia, and this association was significant (odds ratio = 9.96; p = 0.006). CONCLUSION: Subjects with TE have also a risk for lifestyle-related disease as well as the general Japanese population. In addition, cervical spine radiography and magnetic resonance imaging are recommended to assess block vertebrae in subjects with TE with gallbladder aplasia who develop shoulder pain.

Increased serum leukocyte cell-derived chemotaxin 2 (LECT2) levels in obesity and fatty liver.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a signaling molecule expressed in the liver and regulated by Wnt/ß-catenin pathways implicated in hepatic metabolism. However, the clinical relevance of LECT2 in obesity and fatty liver is unknown. The objective of this study was to determine whether serum LECT2 levels are affected by of obesity and fatty liver. A cross sectional study comprising 231 Japanese adult subjects were tested for LECT2 using a highly sensitive assay. We evaluated the associations between LECT2 and the anthropometric or clinical markers of obesity and fatty liver. The mean serum LECT2 levels were 43.5 ± 13.6 ng/mL. LECT2 positively correlated with all the anthropometric measures of obesity: body mass index, waist circumference, waist-to-hip ratio, and waist-to-height ratio (W/Ht). Multiple regression analysis revealed that LECT2 is independently related to γ-glutamyl transpeptidase (γ-GTP), triglyceride, and age in males, whereas in females it was related to the homeostasis model assessment ratio, blood urea nitrogen, high-density lipoprotein cholesterol, and γ-GTP. Receiver operating characteristics curve analyses revealed that LECT2 correlated with obesity [area under the curve (AUC) 0.655, 95% confidence interval (CI) = 0.551-0.758, p = 0.002 in males; AUC 0.670, 95% CI = 0.570-0.770, p < 0.001 in females] and fatty liver (AUC 0.646, 95% CI = 0.544-0.749, p = 0.004 in males; AUC 0.733, 95% CI = 0.621-0.844, p < 0.001 in females). The present study indicates that serum LECT2 levels are increased by obesity and fatty liver, and suggests that LECT2 is a novel obesity-related protein.

Nonalcoholic fatty liver is a risk factor for postprandial hyperglycemia, but not for impaired fasting glucose.

PURPOSE: The first aim of this study was to elucidate the relationship between impaired glucose tolerance (IGT) and nonalcoholic fatty liver. The second was to make a rule regarding to whom 75-g oral glucose tolerance tests (OGTTs) should be applied to identify subjects with IGT and diabetes mellitus (DM) in the annual check-up at the human dry dock. METHODS: A total of 716 subjects who visited the Department of General Medicine of the International Medical Center of Japan from May 2001 through January 2008 for an annual check-up at the human dry dock were analyzed. We evaluated risk factors related to nonalcoholic fatty liver using multivariate logistic regression analysis and compared the difference of body mass index (BMI) and glucose level at 75-g OGTT at two different time points in subjects whose fatty change had improved or worsened. RESULTS: Nonalcoholic fatty liver was strongly related to 2-h- and 1-h-post-challenge glucose level (P<0.0001 and P=0.018, respectively), but not fasting plasma glucose (FPG) (P=0.706). The risk factors for IGT were nonalcoholic fatty liver (P<0.05), low levels of high-density lipoprotein cholesterol (HDL-C) (P=0.026) and age (P=0.013). A clearly positive relationship was observed between the difference of BMI and 2-h-post-challenge glucose level among the subjects whose fatty change had improved or worsened (R=0.6, P=0.018). CONCLUSIONS: Nonalcoholic fatty liver was clearly related to the 2-h- or 1-h-post-challenge glucose level, but not to FPG, in 75-g OGTT, and this IGT was corrected by body weight reduction in accordance with diminished nonalcoholic fatty liver. Thus, 75-g OGTT should be applied to subjects with nonalcoholic fatty liver to evaluate IGT.

An analysis of risk factors for asymptomatic cerebral infarction.

OBJECTIVE: The aim of this study is to identify risk factors for asymptomatic cerebral infarction (ACI) in the general Japanese population. MATERIALS AND METHODS: A total of 634 subjects (272 men aged 55.4+/-8.8 years and 362 women aged 55.2+/-8.5 years) who visited the Health Management Center at Aoyama Hospital (Tokyo, Japan) from January 2004 through January 2005 for an annual brain dry dock examination were analyzed. We evaluated 21 risk factors for ACI by multivariate logistic regression analysis. RESULTS: Abnormal or potentially abnormal conditions were detected in 258 subjects (40.7% of all subjects who had an annual check-up program for brain disease). The most frequent abnormal finding was ACI, which was observed in 208 subjects. The significant risk factors for ACI, as determined by multivariate logistic analysis, were age (P <0.01), hypertension (P <0.01), and hypertensive vascular changes in the fundus (P <0.05). CONCLUSION: The hypertensive vascular abnormalities in the fundus might be a risk factor for ACI independent of age and hypertension.