Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

A novel method for chronic measurement of pleural pressure in conscious rats.

Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals. Previous attempts to measure pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal. These problems have been eliminated by developing a novel surgical procedure for placement of a pressure-sensitive catheter beneath the pleural surface. The catheter (attached to a radiotelemetry transmitter) is surgically implanted beneath the serosal layer of the esophagus within the thoracic cavity. This is accomplished by making a small incision in the serosal layer of the esophagus caudal to the diaphragm and advancing the catheter cranially into the thoracic cavity until pressure changes are maximal. The accuracy of these measurements was verified by comparison with direct pleural pressure measurements over the range of -3 to -34 cm H2O. The pleural pressure changes remained constant for at least 14 weeks following surgery, and there was no evidence of tissue damage or growth around the catheter. This novel method for measuring pleural pressure chronically in conscious rats will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory (breathing) patterns and lung function in conscious animals.

Preclinical evaluation of recombinant human interleukin-4.

Recombinant human IL-4 (rhuIL-4) is primate-specific and produces multiple biologic effects on lymphoid cells involved in protection against cancer. RhuIL-4 was evaluated in the cynomolgus monkey to support clinical studies for the immunotherapy of cancer. Administration of rhuIL-4 to monkeys by SC injection of 0, 0.5, 2.5 or 12.5 micrograms/kg BID for one-month (with two-week recovery) resulted in alterations in clinical chemistry and hematology (CCH) parameters consistent with a consumptive coagulopathy. Histomorphologic evaluation revealed increased granulopoiesis, testicular atrophy, and proliferative and inflammatory vascular lesions (VL). IVL principally affected the arterial tree with some proliferation of medial smooth muscle. During the latter part of the treatment and recovery period. CCH parameters approached or returned to pretreatment values, the former finding attributed to the production of antibody to rhuIL-4. At final necropsy, bone marrow appeared normal, and IVL decreased in incidence and severity. ELISA studies of serum indicated 50-90% of the monkeys developed antibody titers > 1000 by Day 22 (not observed in man). The frequency and severity of adverse effects due to rhuIL-4 in the clinic appear to be does-related and reversible with few objective responses to therapy observed. Common toxicities included milk to moderated fever and fatigue and an occasional change in hematopoietic, hepatic and renal function. The monkey predicted hematologic findings, but not all target organ effects.

Phagocytosis, killing, and oxidant production by bovine monocyte-derived macrophages upon exposure to Brucella abortus strain 2308.

Phagocytosis and intracellular survival of Brucella abortus, and oxidant production by monocyte-derived macrophages from ten B. abortus-naive cows were studied. Phagocytosis of bacteria opsonized with naive-autologous sera or reactor serum was significantly less than phagocytosis of bacteria opsonized with fetal bovine serum. After phagocytosis, intracellular survival of bacteria opsonized with naive-autologous or reactor sera was significantly less than survival of bacteria opsonized with fetal bovine serum. Production of oxidant by macrophages stimulated with B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera was not significantly different. Although macrophages from one animal showed significantly less phagocytic activity, intracellular killing and oxidant production by macrophages from the ten individual cows toward B. abortus opsonized with naive-autologous, reactor, and fetal calf sera were homogeneous. The abilities of the macrophages to phagocytize and to kill B. abortus were not associated with each other or with oxidant production. Innate resistance or sensitivity to B. abortus was not identified in the cows based on macrophage function.

Comparison of oxidant production by bovine neutrophils and monocyte-derived macrophages stimulated with Brucella abortus strain 2308.

Oxidant production by bovine monocyte-derived macrophages and neutrophils was compared after stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ), and B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera. Neutrophils responded more rapidly to all stimuli and produced up to 100-fold greater oxidant than did equal numbers of bovine monocyte-derived macrophages. Macrophages and neutrophils stimulated with PMA, OZ, and reactor-opsonized B. abortus had higher mean oxidant production than phagocytes exposed to B. abortus opsonized with autologous sera, fetal bovine serum, or nonopsonized bacteria. Stimulation of macrophages by opsonized zymosan, buffer, and B. abortus opsonized with autologous sera, reactor serum, or fetal bovine serum resulted in low levels of oxidant production that were not significantly different. Only PMA caused a significantly higher level of oxidant production by macrophages.

Correlation between anion gap, blood L lactate concentration and survival in horses.

Blood L lactate concentration and anion gap were measured in 32 horses suspected of having metabolic acidosis. There was good linear correlation between these variables (r = 0.90791, P less than 0.0001) and both were good prognostic indicators. Anion gap was a good indicator of the presence but not the severity of L lactic acidosis and was a slightly better prognostic indicator. The ability to predict survival was not improved by the measurement of L lactate in addition to anion gap.

Effect of age on anion gap in clinically normal Quarter Horses.

Anion gap [AG = (Na + K) - (Cl + HCO3)] was determined in Quarter Horses: 10 clinically normal foals 0.5 to 3 days, 2 to 3 weeks, and 5 to 7 weeks of age; 10 yearlings; and ten 2-year-olds. Mean AG (+/- 1 SD) were 12.8 +/- 3.3, 15.2 +/- 2.9, 14.0 +/- 1.6, 12.3 +/- 1.8, and 10.4 +/- 1.2 mEq/L, respectively. The 4 to 5 mEq/L higher AG in 2- to 7-week-old foals, when compared with that in 2-year-old horses, can be explained in part by hyperphosphatemia (2 mEq/L) and lower calcium values (0.4 mEq/L). Decreased globulin concentrations may also contribute to the higher AG. The AG can be useful in the diagnosis and prognosis of metabolic imbalances in the horse. The age of the animal was important in the interpretation of AG. Age adjustment of reference intervals will increase the usefulness of AG in foals.

Ultrastructure of canine toxic neutrophils.

Turpentine-induced inflammation was used as a model to study the ultrastructure of canine toxic neutrophils. Circulating toxic neutrophils contained large amounts of rough endoplasmic reticulum, but lamellar aggregates (Döhle bodies) were not observed. Cytoplasmic basophilia in Wright's stained neutrophils related to the persistence of rough endoplasmic reticulum and polyribosomes. Foamy vacuolation of the cytoplasm at the light microscopic level corresponded with irregular, electron-lucent areas which were not membrane bound. However, the presence of membrane remnants and myelin figures within lucent areas, as well as dilation and rupture of membranous structures in bone marrow precursors, indicated that these areas may have been membrane bound. In severely affected cells, there were few lysosomal granules. Early and intermediate neutrophils in the bone marrow of turpentine-injected dogs contained dilated and fragmented rough endoplasmic reticulum associated with irregular, electron-lucent areas in the cytoplasm and perinuclear space. Myelin figures were often in association with these electron-lucent areas. These findings indicated that cytoplasmic immaturity and cellular degeneration were involved in the morphogenesis of toxic neutrophils in the dog.

Effect of age on liver enzyme activities in serum of healthy quarter horses.

Serum alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), 5'nucleotidase (5'NT), sorbitol dehydrogenase (SDH), and aspartate transaminase activities were measured in 10 clinically healthy foals, 10 yearlings, and 10 two-year-old Quarter Horses. Enzyme activities in foals at 0.5 to 3 days, 2 to 3 weeks, and 5 to 7 weeks of age were compared with enzyme activities from yearling and 2-year-old horses. Multivariate analyses of variance revealed significantly higher enzyme values in foals (P less than 0.002). This increase was mainly a result of higher ALP and GGT activities, with lesser effects due to higher SDH and 5'NT activities. Standard deviations for ALP and GGT were also larger in foals than in adult horses. The wide variation of ALP and GGT activities may limit their usefulness in the diagnosis of hepatic disease in foals. Standard deviations for serum AST, SDH, and 5'NT activities were smaller. These enzymes may be indicators of hepatobiliary disease in foals. The high serum enzyme activities in healthy foals may reflect a physiologic difference between foals and adult horses. Relative hepatic mass (as a percentage of body weight) and enzyme activity per gram of hepatic tissue are high in young animals, indicating that the high serum enzyme activities in foals are due partly to a high rate of enzyme production and release.

Blood biochemical response to sodium bicarbonate infusion during sublethal endotoxemia in ponies.

Hypertonic NaHCO3 infusion caused blood volume expansion, increased blood bicarbonate concentration, and delayed the onset of hypophosphatemia in ponies with endotoxemia. However, NaHCO3 infusion did not normalize blood pH, and it increased blood L-lactate concentration, and caused hypokalemia, hypernatremia, and hyperosmolality. The deleterious effects of NaHCO3 infusion in endotoxemia ponies outweighed the beneficial effects. The role of hypertonic NaHCO3 given IV for treatment of endotoxemia in equids must be reevaluated.

Effect of acute acidemia on blood biochemical variables in healthy ponies.

L-Lactic acid and D,L-lactic acid infusion in ponies resulted in metabolic acidosis with high anion gap (AG). Increased AG was explained entirely by increased blood L- and D-lactate concentrations. Hydrochloric acid infusion caused metabolic acidosis with decreased AG. Saline (NaCl) infusion caused mild metabolic acidosis, with no significant change in AG. Plasma K+ concentration was decreased by all types of infusions, with a maximum of 0.50, 0.25, 0.40, 0.50 mmol/L below baseline at the end of infusion in the L-lactic acid-, D,L-lactic acid-, HCl-, and NaCl-infused ponies, respectively. Only hydrochloric acid had a tendency to increase plasma K+ concentration. Hypophosphatemia developed in NaCl- and HCl-infused ponies, but not in the D,L-lactic acid-infused ponies. Serum inorganic phosphate concentration in L-lactic acid-infused ponies increased initially, but was significantly (P less than 0.05) lower than values in the other ponies at 4 hours after onset of infusion. In ponies, the effect of acidemia on plasma K+ and serum inorganic phosphate concentrations was similar to that reported for other species. Changes were small in magnitude and depended on the nature of the acid anion. Results indicate that large changes in plasma K+ and serum inorganic phosphate concentrations during acidosis are probably not a direct result of acidemia.

Clinical, clinicopathologic, and parasitologic observations of trypanosomiasis in dogs infected with North American Trypanosoma cruzi isolates.

Nineteen purebred Beagles of various ages (4, 5, 13, and 47 weeks) were inoculated with North American Trypanosoma cruzi isolates obtained from an opossum (Tc-O), an armadillo (Tc-A), or a dog (Tc-D). Dogs were grouped on the basis of clinical outcome of infection. During the acute stage of disease, dogs of group 1 (n = 7 inoculated with Tc-O or Tc-A) died or were euthanatized because of the severity of disease. Dogs of group 2 (n = 5 inoculated with Tc-O or Tc-A) developed acute disease, but survived to develop chronic disease. Dogs of group 3 (n = 7 Tc-D-inoculated dogs) developed neither acute nor chronic disease. Dogs of group 4 (n = 4--2 dogs 13 weeks old and 2 dogs 47 weeks old) served as noninoculated controls. Clinical signs associated with severe acute myocarditis developed in dogs of groups 1 and 2 between postinoculation day (PID) 15 and 28. Generalized lymphadenopathy and lymphocytosis were observed in all dogs of groups 1, 2, and 3 between PID 14 and 17. Serum alanine transaminase and aspartate transaminase activities and urea nitrogen concentration were high, and glucose concentration was low prior to death of dogs in group 1. Serum activities of isoenzymes of creatine kinase were significantly (P less than 0.05) high in only 1 dog (group 1), whereas serum lactate dehydrogenase isoenzyme activities were not significantly high in any dog.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypertonic vs isotonic saline solution on responses to sublethal Escherichia coli endotoxemia in horses.

Cardiovascular responses to sublethal endotoxin infusion (Escherichia coli, 50 micrograms/ml in lactated Ringer solution at 100 ml/h until pulmonary arterial pressure increased by 10 mm of Hg) were measured 2 times in 5 standing horses. In a 2-period crossover experimental design, horses were either administered hypertonic (2,400 mosm/kg of body weight, IV) or isotonic (300 mosm/kg, IV) NaCl solution after endotoxin challenges. Each solution was administered at a dose of 5 ml/kg (infusion rate, 80 ml/min). Complete data sets (mean arterial, central venous, and pulmonary arterial pressures, pulmonary arterial blood temperature, cardiac output, total peripheral vascular resistance, heart rate, plasma osmolality, plasma concentration of Na, K, Cl, and total protein, blood lactate concentration, and PCV) were collected at 0 (baseline, before endotoxin infusion), 0.25, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 hours after initiation of the endotoxin infusion. Blood constituents alone were measured at 0.5 hour and cardiovascular variables alone were evaluated at 0.75 hour. By 0.25 hour, endotoxin infusion was completed, a data set was collected, and saline infusion was initiated. By 0.75 hour, saline solutions had been completely administered. Mean (+/- SEM) cardiac output decreased (99.76 +/- 3.66 to 72.7 +/- 2.35 ml/min/kg) and total peripheral resistance (1.0 +/- 0.047 to 1.37 +/- 0.049 mm of Hg/ml/min/kg) and pulmonary arterial pressure (33.4 +/- 0.86 to 58.3 +/- 1.18 mm of Hg) increased for both trials by 0.25 hour after initiation of the endotoxin infusion and prior to fluid administration. For the remainder of the protocol, cardiac output was increased and total peripheral resistance was decreased during the hypertonic, compared with the isotonic, saline trial.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypovolemia, hyperosmolality, and acidosis associated with intraperitoneal infusion of nitrofurazone solution in healthy horses.

Nitrofurazone solution containing 0.2% nitrofurazone and 99.8% polyethylene glycol was given to 4 healthy horses (2 L in 2 L of lactated Ringer solution, intraperitoneally). Horses developed hypovolemia, hyperosmolality, and mixed respiratory and metabolic acidosis. These changes were largely attributable to polyethylene glycol, but a contribution of nitrofurazone cannot be excluded. Intraperitoneal infusion of nitrofurazone solution in horses is contraindicated.

Evaluation of gamma-glutamyl transpeptidase-to-creatinine ratio from spot samples of urine supernatant, as an indicator of urinary enzyme excretion in dogs.

gamma-Glutamyl transpeptidase activity was measured accurately in canine urine supernatant without gel filtration and was relatively stable at 4 C for at least 4 days after collection. The urinary gamma-glutamyl transpeptidase-to-creatinine ratio in spot samples was simple and quick to measure and was correlated with the 24-hour enzyme excretion. However, the usefulness of this ratio may be limited by within-day variation, and a questionable theoretical basis for its validity.

Quantitative urinalysis in kittens from four to thirty weeks after birth.

To evaluate renal function and obtain reference values for measurements of urinary excretion of various substances, quantitative urinalysis was performed in healthy, growing kittens from 4 to 30 weeks after birth. Endogenous creatinine clearance, 24-hour urine protein excretion, and urine protein-to-creatinine ratio were determined. Additionally, fractional excretion to creatinine clearance was calculated for calcium, inorganic phosphorus, sodium, potassium, and chloride. Mean +/- SD endogenous creatinine clearance values (range, 3.80 +/- 0.48 to 4.74 +/- 0.61 ml/min/kg) were significantly (P less than 0.0001) higher in kittens 9 to 19 weeks old, compared with younger (range, 1.39 +/- 0.85 to 3.59 +/- 0.86 ml/min/kg) and older kittens (range, 2.69 +/- 0.40 to 3.46 +/- 0.37 ml/min/kg). Mean values for all kittens for 24-hour urine protein excretion (range, 2.54 +/- 1.81 mg/kg at 4 weeks to 11.39 +/- 7.61 mg/kg at 14 weeks) and for urine protein-to-creatinine ratio (range, 0.14 +/- 0.03 to 0.34 +/- 0.18) varied from week to week of age. The urine protein-to-creatinine ratio in kittens greater than or equal to 9 weeks old correlated well (R2 = 0.861) with 24-hour urine protein excretion. Urinary fractional excretion of calcium, inorganic phosphorus, sodium, potassium, and chloride in kittens varied among age groups, being significantly (P less than 0.01) different for potassium and calcium in young kittens (4 to 6 weeks) and older kittens (greater than or equal to 7 weeks).

Evaluation of a technique for measurement of gamma-glutamyltranspeptidase in equine urine.

gamma-Glutamyltranspeptidase (GGT) activity in equine urine was measured, using an assay developed for use with serum and was found to be reproducible. The GGT activity was measured in samples prepared by serial dilution of exogenous GGT with equine urine, and the activity was determined to be linear between 21 IU/L and 407 IU/L. The behavior of exogenously added GGT was compared in equine serum and urine. The enzyme behaved similarly in both fluids. The GGT activity was measured in serum and urine samples after storage at -20, 4, and 25 C for 24 and/or 72 hours. Enzyme activity decreased after storage at all temperatures and times, but the decrease was greatest and most variable at -20 C. The urine GGT: urine creatinine ratio was calculated on serial urine samples collected over 24 hours. Although the urine GGT activity and creatinine concentration had significant variation, the ratio remained constant. The urine GGT: urine creatinine ratio was calculated from random urine samples collected from 27 clinically healthy adult horses and was found to be 10.52 +/- 4.78 (mean +/- SD).

Urinary gamma-glutamyl transpeptidase activity in dogs with gentamicin-induced nephrotoxicity.

Serum creatinine concentrations, 24-hour endogenous creatinine clearance, and 24-hour urinary gamma-glutamyl transpeptidase (UGGT) activity were measured daily in 6 dogs given nephrotoxic dosages of gentamicin (10 mg/kg of body weight) every 8 hours for 10 days. Mean UGGT activity was significantly increased by day 5 (P less than 0.05) and preceded significant increases in serum creatinine values (greater than 2.0 mg/dl) observed on day 9. Endogenous creatinine clearance remained within normal limits (2.98 +/- 0.96 ml/min/kg) until day 8. Urinalyses performed 8 days after initiation of gentamicin treatment indicated renal tubular damage (granular casts) in 1 of the 6 dogs, and glucosuria in 3 of the 6 dogs. Measurement of UGGT activity was a more sensitive and reliable method of assessing acute renal tubular damage induced by gentamicin than were serum creatinine concentrations or 24-hour endogenous creatinine clearance.

Contribution of whole blood L-lactate, pyruvate, D-lactate, acetoacetate, and 3-hydroxybutyrate concentrations to the plasma anion gap in horses with intestinal disorders.

Increased anion gap (AG) was due, in part, to L-lactic acidosis in 14 of 14 horses with intestinal disorders. In a few horses, increased whole blood concentrations of D-lactate made a minor contribution to the AG. However, the increase in AG was often greater than the sum of the increases in these 2 acid anions. This unexplained increase was not a result of increases in whole blood pyruvate, 3-hydroxybutyrate, or acetoacetate concentrations or serum albumin or phosphate concentrations. Identification of other anions causing increased AG could lead to better understanding, diagnosis, and treatment of metabolic imbalances in critically ill horses.

Comparison of single-dose and conventional trimethoprim-sulfadiazine therapy in experimental Staphylococcus intermedius cystitis in the female dog.

Cystitis was produced in 4 groups of 6 female dogs each, using salicylic acid, ethanol, and Staphylococcus intermedius. Group-I dogs served as nontreated controls. Starting 2 days after infection was induced, group-II dogs were treated with trimethoprim-sulfadiazine at a dosage of 15 mg/kg given orally 2 times a day for 21 days; groups-III and -IV dogs were treated with single oral dosages of the antibiotic at 60 mg/kg and 90 mg/kg, respectively. Group-I dogs (controls) remained infected for the 26-day duration of the study. The response to therapy seen in group-II dogs was better than the therapeutic responses in groups-III and -IV dogs (P less than 0.05). Results of the present study do not support the efficacy of single-dose therapy for this model of cystitis.