Antimonocyte chemoattractant protein-1 gene therapy reduces experimental in-stent restenosis in hypercholesterolemic rabbits and monkeys.

In-stent restenosis results exclusively from neointimal hyperplasia due to mechanical injury and a foreign body response to the prosthesis. Inflammation mediated by monocyte chemoattractant protein-1 (MCP-1) might therefore underlie in-stent restenosis. We recently devised a new strategy for anti-MCP-1 gene therapy by transfecting an N-terminal deletion mutant of the MCP-1 gene into skeletal muscles. We used this strategy to investigate the role of MCP-1 in experimental in-stent restenosis in hypercholesterolemic rabbits and monkeys. Transfection of the mutant MCP-1 gene suppressed monocyte infiltration/activation in the stented arterial wall and markedly reduced the development of neointimal hyperplasia. This strategy also suppressed local expression of MCP-1 and inflammatory cytokines. Therefore, inhibition of MCP-1-mediated inflammation is effective in reducing experimental in-stent restenosis. This strategy might be a useful form of gene therapy against human in-stent restenosis.

Fibromuscular cap composition is important for the stability of established atherosclerotic plaques in mature WHHL rabbits treated with statins.

We examined the relationship between plaque vulnerability and fibromuscular cap composition using hydrophilic pravastatin and lipophilic fluvastatin. WHHL rabbits aged 10 months were given pravastatin (50 mg/kg) or fluvastatin (20 mg/kg) for 52 weeks. The atherosclerotic lesions were immunohistochemically or conventionally stained and the components were analyzed with a color image analyzer. Compared with the control group, the plasma cholesterol levels were decreased by about 25% in both statin groups. Pravastatin decreased the lipid components (macrophages+extracellular lipids) in whole aortic plaques by 34% and the fibrous caps of coronary plaques by 55%. Fluvastatin decreased the fibromuscular components (smooth muscle cells+collagen fibers) in whole aortic plaques and in the fibromuscular caps of the aortic and coronary plaques. In the pravastatin group, the vulnerability index, the ratio of (lipid components)/(fibromuscular components), was decreased in whole aortic plaques by 28% and in the fibromuscular caps of coronary lesions by 61%, while the indexes were increased in the fluvastatin group. The incidence of vulnerable plaques was decreased by 74% in the coronary plaques of the pravastatin group. Our results suggest that the stability of atheromatous plaques was improved due to a decrease of the lipid components and vulnerability index of the fibromuscular cap by pravastatin.

Statins alter smooth muscle cell accumulation and collagen content in established atheroma of watanabe heritable hyperlipidemic rabbits.

BACKGROUND: Acute coronary syndromes often result from rupture of vulnerable plaques. The collagen content of plaques probably regulates their stability. This study tested whether HMG-CoA reductase inhibitors (statins) alter interstitial collagen gene expression or matrix metalloproteinase (MMP) levels in rabbit atheroma. METHODS AND RESULTS: We administered equihypocholesterolemic doses of pravastatin (a hydrophilic statin, 50 mg. kg(-1). d(-1), n=9), fluvastatin (a cell-permeant lipophilic statin, 20 mg. kg(-1). d(-1), n=10), or placebo (n=10) to mature Watanabe heritable hyperlipidemic rabbits for 52 weeks. The fluvastatin group achieved a much higher peak plasma concentration (23.7 micromol/L) than did the pravastatin group (1.3 micromol/L) under these conditions. Immunohistochemistry revealed that MMP-1, MMP-3, and MMP-9 expression by macrophages in the intima was lower in both the pravastatin and fluvastatin groups than in the placebo group, whereas there was no difference in macrophage numbers. Numbers of intimal smooth muscle cells (SMCs) (identified by immunohistochemistry) and expression of type I procollagen mRNA (detected by in situ hybridization), however, were significantly higher in the pravastatin group than in the fluvastatin group. Treatment with pravastatin, but not fluvastatin, preserved interstitial collagen content in vivo (detected by picrosirius red polarization). In vitro, fluvastatin, but not pravastatin, decreased numbers of rabbit and human aortic SMCs without altering procollagen I mRNA expression. CONCLUSIONS: This study showed that statins can reduce MMP expression in atheroma and that cell-permeant statins can decrease SMC number and collagen gene expression in vivo.

Stability of atheromatous plaque affected by lesional composition: study of WHHL rabbits treated with statins.

We examined how lesional composition is related to the stability of coronary plaques. WHHL rabbits (12 months old) were given pravastatin and fluvastatin orally for 52 weeks. Both statins decreased plasma cholesterol levels by about 25%, but the suppressive effects on the degree of coronary plaques were mild. Macrophage (Mphi) contents in fibromuscular cap regions were decreased by pravastatin, and smooth muscle cell (SMC) contents in those were decreased by fluvastatin. The plaque vulnerability index was low in Mphi)-poor plaques, but high in SMC-poor plaques. Our results suggest that reduction in M(phi)s in the fibromuscular cap is related to plaque stabilization and that reduction in SMCs in the fibromuscular cap is related to plaque destabilization.

Carcinogenicity test of polyoxyethylene(10) nonylphenyl ether (NP-10) in female B6C3F1 mice.

A carcinogenicity study of polyoxyethylene(10)nonylphenyl ether (NP-10) to B6C3F1 mouse was performed using 50 females per group of 4 study groups, or 200 mice in total. Diets containing NP-10 at 0, 500, 1500 and 4500 ppm were prepared and orally administered to the animals repeatedly for 104 weeks, and observation of general conditions, body weight analysis, food consumption analysis, hematologic examination, organ weight analysis and pathological examination were performed. The results are summarized as follows. The mean intake of the test substance in the 500, 1500 and 4500 ppm groups for 104 weeks was 81.5, 254 and 873 mg/kg/day, respectively. There were no differences observed in mortality among the groups and the mortality did not exceed the background data in any groups. There were no signs attributable to the administration of the test substance, and various signs which increased in occurrence with aging were observed in all groups at a similar frequency. Body weight gain was suppressed only in the 4500 ppm group throughout the entire administration period. Food consumption was increased in all treated groups around the early stage of administration and, thereafter, in the 1500 and 4500 ppm groups until the mid-stage of administration. Decreased food efficiency was observed in the 4500 ppm group alone. As a result of the hematologic examination, no changes attributable to the administration of the test substance were observed in any groups. As a result of the organ weight analysis, lower absolute weights of the liver and kidney and higher relative weights of the brain, liver and kidney, which were considered to be changes accompanying the suppressed body weight gain, were observed in the 4500 ppm group. The pathological examination revealed no marked changes in the gross findings in the treated groups. As a result of the histological examination, there were no neoplastic or non-neoplastic lesions in the treated groups which were unequivocally observed to have increased in occurrence. As the above findings show, NP-10 did not cause any increase in the incidence of neoplastic lesions in the mouse by oral administration for 2 years at 873 mg/kg/day (4500 ppm) and was determined to have no carcinogenicity.

Oral chronic toxicity and carcinogenicity test of polyoxyethylene(10)nonylphenyl ether (NP-10) in female F344 rats.

Chronic toxicity and carcinogenicity of polyoxyethylene(10)nonylphenyl ether (NP-10) to Fischer 344 rats were investigated using 70 females per group in 4 study groups, or 280 rats in total. Diets containing NP-10 at 0, 1000, 3000 and 9000 ppm were prepared and orally administered to the animals repeatedly for 52 weeks for a chronic toxicity study and for 104 weeks for a carcinogenicity study. Observations of general condition, body weight analysis, food consumption analysis, hematologic examination, blood chemistry examination (only at Week 52 of administration), urinalysis (only at Week 52), ophthalmologic examination (immediately prior to administration and at Week 52), organ weight analysis and pathological examination were performed. The results are summarized as follows. The mean intake of the test substance was 60.5, 182 and 559 mg/kg/day in the chronic toxicity study for 52 weeks and 55.2, 166 and 520 mg/kg/day in the carcinogenicity study for 104 weeks in the 1000, 3000 and 9000 ppm groups, respectively. Mortality decreased approximately in a dose-related manner, with 28% in the control group, 26% in the 1000 ppm group, and 14% each in the 3000 and 9000 ppm groups. In general condition, there were no signs attributed to the treatment with NP-10. Body weight gain was suppressed in the 9000 ppm group throughout the administration period and in the 3000 ppm group during Weeks 21-88. Food consumption decreased in the 9000 and 3000 ppm groups. Food efficiency was lower in the 9000 and 3000 ppm groups. As a result of the hematologic examination, hematocrit value, hemoglobin value, red blood cell count, platelet count and MCV were lower and MCH and MCHC higher in the 9000 ppm group at Week 52 of administration. At Week 104, the neutrophil ratio was higher and lymphocyte ratio lower in the 3000 and 9000 ppm groups, and furthermore, hematocrit value, hemoglobin value, MCV and MCH were slightly lower in the 9000 ppm group. In the blood coagulability tests, prothrombin time was slightly shortened in the 9000 ppm group at Week 52. As a result of the blood chemistry examination, total protein and albumin values were higher and total bilirubin, uric acid and trygliceride value lower in the 3000 ppm and higher dose groups. Furthermore, the free cholesterol value was higher and the values of potassium, cholesterol ester ratio, GOT, GPT, ALP and cholinesterase were lower in the 9000 ppm group. As a result of the urinalysis, the specific gravity of urine was higher and urine pH acidic in some animals. As a result of the ophthalmologic examination, no abnormal animals were found in the 9000 ppm group. As a result of the organ weight analysis, absolute and relative weights of the liver and adrenals were higher in the 3000 and/or 9000 ppm groups as changes which were considered attributable to the test substance and, in addition, organs with a lower absolute weight and higher relative weight with the suppressed body weight gain were observed in the 9000 ppm group. The histopathological examination revealed no marked findings in necropsy observation or histology in the treated groups in the animals killed at Weeks 52, 104 as well as those killed moribund and dead animals. In the histological findings, bile duct hyperplasia of liver in the animals killed at Week 52, proliferative duct of pancreas in the animals killed at Week 104, pigment of deposit in pituitary and angiectasis of adrenals in the animals killed at moribund and dead animals were observed in a slightly larger number in the treated groups, but none of these changes were different in degree from the control and were not considered to be specific lesions. As a result of the overall study of the neoplastic lesions of all animals killed on schedule and of moribund and dead animals, no tumors were found in the treated groups which had increased in occurrence. Based on the above findings, it was determined that the no-adverse-effect level in the chronic toxicity study was 1000 ppm (

[Effects of mesalazine on liver carcinogenesis in medium-term bioassay using rats].

A two stage carcinogenesis promotion test using phenobarbital (PB) as a positive control was performed on mesalazine in rats (F344,male). Pathological and immunohistological examinations were performed to examine the cell damage and proliferation in the liver and kidneys. As the initiation treatment, groups 1,2,3 and 5 were administered 300 mg/kg diethylnitrosamine (DEN)dissolved in 0.9% physiological saline, and group 4 was administered 5 ml/kg 0.9% physiological saline once intraperitoneally. Then group 1 was orally administered a water solution (5 ml/kg) containing 0.5% CMC-Na, and groups 2,3 and 4 similar water solution but containing 150, 300 and 300 mg/kg mesalazine, respectively. Group 5 was administered 0.05% PB mixed in feed from weeks 2 to 8. Partial (2/3) hepatectomy was performed in all 5 groups at week 3 after DEN administration. NO clear differences between the groups were observed in general conditions, body weight or amount of food consumption. The number or area-size of hepatic GST-P positive altered cell foci revealed no significant differences between groups 1,2 and 3, but a significant increase in number and area-size was observed in group 5. No GST-P positive cell foci were detected in group 4. The number of altered cell foci (H.E. staining) in the DENgroups administered mesalazine was the same as that in group 1. Thus, mesalazine did not promote hepatocarcinogenesis in the present experimental system. Statistically insignificant appearances of basophilic and acidophilic changes were observed in the renal tubular epithelium and mineral deposits in the renal papillary region and cortical margin region. The PCNA labeling rate was significantly lower in group 4, corresponding with the histological finding showing no proliferation of the renal tubular epithelium. Judging from the above test results, mesalazine was likely to show neither a promotion effect on the initiation induced by DEN nor cell proliferative activity on the kidneys by administration for this experimental period.

A case of multiple osteochondroma in the rat.

The occurrence of spontaneous osteochondroma in rats is extremely rare and only a few cases have been reported. Spontaneous multiple osteochondroma was found in a male SD strain rat, at the age of 58 weeks from the control group in a toxicity study. Histological findings of both a walnut-sized mass involving the humerus and scapula of right forelimb and a soybean-sized mass adjacent to the costochondrial junction of the right posterior rib were similar in appearance. The outer layer of the tumors consisted of hyaline cartilage and the core of the tumors consisted of trabecular bone with abundant fatty bone marrow. The periosteum of the surface was continuous with that of the parent bone, and cortical bone and the medullary cavity of the parent bone communicated with those of the tumors. Because of showing progressive enlargement, multiple osteochondromas in the rat were considered to be neoplastic in nature.

Preventive effect of betamipron on nephrotoxicity and uptake of carbapenems in rabbit renal cortex.

The preventive effect of betamipron (N-benzoyl-3-propionic acid: BP) on the renal uptake and nephrotoxicity of carbapenems (panipenem and imipenem) was studied in rabbits. Panipenem, a new carbapenem antibiotic, induced nephrotoxicity at a dose of 200 mg/kg, i.v., but this was less severe than that caused by a single dose of imipenem or cephaloridine. Along with the significant reduction of nephrotoxicity, the uptake of these carbapenems in the renal cortex was remarkably inhibited by simultaneous treatment with BP (200 mg/kg, i.v.). These results suggest that BP reduces the nephrotoxicity of carbapenems through inhibiting the active transport of carbapenems in the renal cortex. Because of the low toxicity of BP (LD50 in the rat, more than 3,000 mg/kg, i.v.), it was concluded that BP might be a good candidate for reducing the nephrotoxicity induced by panipenem or imipenem.

Protective effect of N-acyl amino acids (NAAs) on cephaloridine (CER) nephrotoxicity in rabbits.

The protective effect of N-acyl amino acids (NAAs) against cephaloridine (CER)-induced nephrotoxicity was studied in rabbits. A large single intravenous dose of CER (more than 100 mg/kg) induced severe proximal tubular necrosis. Simultaneous treatment with several NAAs (at dosages of 100, 200 mg/kg, etc., i.v.), such as N-benzoyl-beta-alanine (NBBA), N-benzoyl-6-aminocaproic acid, and N alpha,epsilon-dibenzoyl-D,L-lysine, remarkably suppressed the histopathological damage in the kidney induced by CER. NAAs have generally low toxicity in laboratory animals (e.g., the LD50 of NBBA was more than 3,000 mg/kg, i.v. in rats), and NAAs were suggested to be good candidates for reducing the nephrotoxicity of CER and other beta-lactam antibiotics.

Historical control data of organ weight and gross findings in F344/DuCrj rats and B6C3F1 mice.

Organ weight and gross postmortem findings of control Fischer 344/DuCrj rats and B6C3F1 mice are presented from subchronic, chronic toxicity and carcinogenicity studies that were conducted over a 9 year period at our center (An-Pyo Center). The mean organ weight of the liver, kidney, spleen and the lung were increased associated with age in both species. The most common findings observed at 109 weeks in both species were thymic atrophy, enlargement of the spleen, dilation of the lumen of the uterus and ovarian cysts. Male and female Fischer 344/DuCrj rats commonly exhibited a granular surface of the kidneys, hypertrophy of and/or nodular pituitary glands and subcutaneous tissue masses. Multiple white patches and hypertrophy or atrophy of the testes, atrophy of the seminal vesicles and prostate and nodules in the pancreas were seen frequently in male rats. Malformative nodule in the liver was a common finding in female rats. The most common lesions seen in both male and female B6C3F1 mice were nodules in the liver and lungs and enlargement of the lymph nodes. Nodules on the preputial gland were commonly seen in the males. Sex differences were evident in the incidence of some of the above findings. These historical data will contribute to analyze the organ weight and gross findings at necropsy in long-term toxicity and carcinogenicity studies.

Heterotopic gastric mucosa of the small intestine in laboratory beagle dogs.

Out of the 365 young laboratory beagle dogs which were used in 17 toxicity bioassays, 15 cases (4.1%) were diagnosed as having congenital heterotopic gastric mucosa of the small intestine. Its incidence in the male dogs (12 cases out of 187) was higher than in the female dogs (3 cases out of 178). Grossly, the lesions were seen as an ulcerous focus of the small intestine, 25 cm to 88 cm proximal to the ileocecal valve. All of the lesions were quite similar histologically and electron microscopically to the normal gastric mucosa, which are composed of the surface mucous cells, chief cells, parietal cells, mucous neck cells and basal granulated cells of the stomach. And consequently, they were considered to be that of a congenital heterotopic tissue in the small intestine. The only morphological characteristic of these lesions different from the regular gastric mucosa was an association with the tubular structure seen in the basal region of these mucosal layers. These cells were considered to be of mucous-secreting cell origin because of secreting type III mucous evident from paradoxical concanavalin A or periodic acid Schiff stains. They seemed to be protecting the surrounding intestinal mucosa from gastric acid.

[Pathological study on nephrocalcinosis in Fischer 344/Du Crj rats].

Histopathological examinations on nephlocalcinosis of the Fischer 344 (F344) rats were carried out. As the results of comparison on its appearance among F344, Wistar and SD strains of rats, F344 female rats showed the most severe nephrocalcinosis. Nephrocalcinosis developed between 4 weeks and 8 weeks and was likely to keep its appearance through 108 weeks of the survival period of the rats. Histologically, mineral deposit was always observed at cortico-medullary junction. It seemed to locate at the outer portion of the basement membrane of the tubular epithelium, adjacent to the capillary wall in the connective tissue. Four weeks after ovariectomy at 4 weeks of age, the rats showed a decrease in degree of nephrocalcinosis. In contrary, the rats treated with estorone following ovariectomy revealed an increase in degree of nephrocalcinosis. It was suggested that the oestrogen-type sex hormone appeared to give a role in nephlocalcinosis.

Electrocardiographic Manifestations in Patients with Uterine Myoma.

Electrocardiographic manifestations in 114 patients with uterine myoma were investigated. Classification of electrocardiograhic patterns was based on the Minnesota Code. No reportable electrocardiogram items were recorded in 24 patients (21.2% of the 114 myoma patients. High-amplitude R waves were found in 23 patients (20.4%), depressed ST segments were recorded in 28 patients (24.8%), and abnormal T waves, such as flat or negative T, were noted in 14 patients (12.4%) of the 114 patients with myoma. Some of these items are explainable by anemia due to excessive uterine hemorrhage in myoma patients. However, it could be concluded that all of these electrocardiographic changes are characteristic of anemic patients. Further studies on cardiovascular disturbances in myoma patients will be needed.