Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.

The endothelial cell-specific vascular endothelial growth factor (VEGF) and its cellular receptors Flt-1 and Flk-1 have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis and the impaired vessel formation in Flk-1 and Flt-1 deficient embryos. However, because Flt-1 also binds placental growth factor, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES) and even more impaired in homozygous VEGF-deficient (VEGF-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F1-VEGF+/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inactivation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.

Physiological consequences of loss of plasminogen activator gene function in mice.

Indirect evidence suggests a crucial role for the fibrinolytic system and its physiological triggers, tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator, in many proteolytic processes. Inactivation of the t-PA gene impairs clot lysis and inactivation of the u-PA gene results in occasional fibrin deposition. Mice with combined t-PA and u-PA deficiency suffer extensive spontaneous fibrin deposition, with its associated effects on growth, fertility and survival.

Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization.

Homozygous plasminogen activator inhibitor-1 (PAI-1)-deficient (PAI-1-/-) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-1 was demonstrated by Southern blot analysis. A 3.0-kb PAI-1-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-1+/+) but not from PAI-1-/- mice. Plasma PAI-1 levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63 +/- 2 ng/ml, 30 +/- 10 ng/ml, and undetectable (< 2 ng/ml) in PAI-1+/+, heterozygous (PAI-1+/-) and PAI-1-/- mice, respectively (mean +/- SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1+/+ but not of PAI-1-/- mice. SDS-gel electrophoresis of plasma incubated with 125I-labeled recombinant human tissue-type plasminogen activator revealed an approximately 115,000-M(r) component with plasma from endotoxin-stimulated (0.5 mg/kg) PAI-1+/+ but not from PAI-1-/- mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAI-1-/- mice were viable, produced similar sizes of litters as PAI-1+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities.

Small animal thrombosis models for the evaluation of thrombolytic agents.

BACKGROUND: Two thrombosis models for the evaluation of thrombolytic agents in small animals (less than 100 g) were evaluated: an iodine-125 fibrin-labeled rat plasma clot in the inferior caval vein of 3-4-week-old rats and a pulmonary embolus in adult hamsters that had been obtained by injection of a 125I fibrin-labeled human plasma clot. The extent of thrombolysis was determined by continuous external monitoring of radioisotope over the thrombus region and by ex vivo recovery of residual clot. METHODS AND RESULTS: In the rat model, infusion of solvent for 60 minutes was associated with mean +/- SEM lysis within 90 minutes of 13 +/- 3% (n = 8) by external counting and 26 +/- 4% (n = 8) by radioisotope recovery. Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) over 60 minutes caused dose-dependent progressive clot lysis; with 0.5 mg/kg, producing a plasma level of 0.14 +/- 0.04 microgram/ml, lysis was 64 +/- 9% (n = 4) by external gamma counting and 78 +/- 4% (n = 4) by residual isotope in the vein segment and was not associated with significant fibrinogen or alpha 2-antiplasmin breakdown. In the hamster model, infusion of solvent for 60 minutes was associated with lysis within 90 minutes of 19 +/- 4% (n = 11) by external gamma counting and 31 +/- 3% (n = 14) by residual radioisotope. Intravenous rt-PA during 60 minutes resulted in dose-dependent progressive thrombolysis; with 0.5 mg/kg, producing a plasma level of 0.14 +/- 0.01 micrograms/ml, lysis was 50 +/- 4% (n = 4) by external gamma counting and 78 +/- 5% (n = 4) by residual radioactivity, without an extensive decrease in fibrinogen or alpha 2-antiplasmin. Parallel experiments in the rabbit jugular vein thrombosis model with a rabbit blood clot with intravenous infusion over 4 hours produced 7 +/- 2% (n = 9) lysis with solvent and dose-dependent progressive lysis with rt-PA; with 1 mg/kg, producing a plasma level of 0.20 +/- 0.03 microgram/ml, lysis was 56 +/- 7% (n = 7) by external gamma counting and 61 +/- 7% (n = 7) by residual radioactivity, without extensive consumption of fibrinogen or alpha 2-antiplasmin. CONCLUSIONS: These two thrombosis models in small animals are as reproducible and quantitative as the extensively used rabbit jugular vein thrombosis model. The hamster pulmonary embolism model is superior because it is simpler and more straightforward and allows the performance of as many as 10 experiments by one investigator in 1 day.

Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model.

A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 microliters 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. Recombinant t-PA (home-made rt-PA or Activase) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in microgram/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1 + e-a(ax-eh] and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = 1/4 ac.eb) were determined. With rt-PA, these parameters were c = 72 +/- 6% (mean +/- SEM), b = 0.19 +/- 0.08 mg/kg, z = 68 +/- 25% lysis per mg/kg, with corresponding values of 87 +/- 5%, 0.07 +/- 0.03 mg/kg and 150 +/- 38% lysis per mg/kg for Activase (p = NS). Deletion of the finger and growth factor domains in rt-PA (rt-PA-delta FE) was not associated with marked alteration of the thrombolytic potency (c = 90 +/- 30%, b = 0.34 +/- 0.35 mg/kg, and z = 54 +/- 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Predictive value of increased plasma levels of fibronectin in gestational hypertension.

Blood pressure, proteinuria, and plasma fibronectin and plasminogen activator inhibitor-1 levels were measured in 120 apparently healthy normotensive primigravid women during the first, second, and third trimesters of pregnancy and 2 days post partum. Thirty-two women developed hypertension (diastolic blood pressure greater than or equal to 90 mm Hg) that in 17 women was associated with proteinuria (greater than 0.3 gm/day). Fibronectin levels were 83% +/- 22% of normal (mean +/- SD) during the first trimester and 75% +/- 20% at term in the healthy women but increased from 94% +/- 36% to 187% +/- 36% in the women who developed gestational hypertension (with or without proteinuria) (p less than 0.0001). Plasminogen activator inhibitor-1 levels increased from 26 +/- 19 ng/ml to 110 +/- 86 ng/ml in healthy women and from 32 +/- 35 ng/ml to 290 +/- 90 ng/ml in hypertensive women (p less than 0.001). Increased levels of fibronectin at 25 to 36 weeks of pregnancy (greater than or equal to mean + 2 SD of the healthy women, or greater than 140%) were found in 31 of the 32 women with gestational hypertension with or without proteinuria and in 5 of the 88 healthy women (sensitivity 96%, specificity 94%). Fibronectin levels increased 3.6 +/- 1.9 weeks earlier than the onset of hypertension and/or proteinuria. Increased levels of plasminogen activator inhibitor-1 at 25 to 32 weeks (greater than or equal to 280 ng/ml) were found in 16 of the 32 women who developed gestational hypertension with or without proteinuria and in 4 of the 88 healthy women (sensitivity 50%, specificity 95%). We conclude that increased fibronectin levels are the best predictor of gestational hypertension with or without proteinuria and that its level in plasma increases several weeks before the development of hypertension.

Pharmacokinetics of single chain forms of urokinase-type plasminogen activator.

The pharmacokinetics of single chain urokinase-type plasminogen activator (scu-PA) were studied in rabbits and squirrel monkeys using three of its molecular forms, Mr 54,000 isolated from human urine (urinary scu-PA), Mr 54,000 isolated from conditioned media of cultured human lung adenocarcinoma cells (CALU-3) (cellular scu-PA) and its Mr 32,000 proteolytic derivative lacking the NH2-terminal 143 amino acids (scu-PA-32k). After bolus i.v. injection in rabbits with 2.5 kg. b.wt., the disappearance rate of all three forms from plasma could be described by a two-compartment disposition model with the following pharmacokinetic parameters: alpha T1/2, 3.2 to 3.9 min; beta T1/2, 15 to 16 min; volume of the central compartment, 170 to 200 ml; total volume of distribution, 660 to 910 ml; and plasma clearance, 18 to 22 ml/min. Similar results were obtained with cellular scu-PA in squirrel monkeys. Plasma euglobulin fibrinolytic activity disappeared in parallel with antigen, suggesting that the primary mechanism of disappearance is via clearance of the intact molecule. The organ distribution of isotope after bolus injection of 125I-labeled scu-PA revealed that clearance occurred via the liver and kidneys in rabbits, but predominantly via the liver in squirrel monkeys. Functional hepatectomy prolonged the alpha T1/2 of scu-PA in rabbits, whereas nephrectomy had no significant effect. Steady-state plasma levels of scu-PA during continuous i.v. infusion were proportional to the dose given, whereas the initial postinfusion disappearance rate of scu-PA from plasma was comparable to that after bolus injection (T1/2 of 5 to 6 min).(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of a modified erythrocyte transketolase assay for assessing thiamine nutritional adequacy.

Transketolase (TK) activity was assessed by incubating hemolyzed whole blood with ribose-5-phosphate both in the presence (TKsat.) and in the absence (TK) of thiamine pyrophosphate (TPP). The amount of pentoses utilized between 5 and 60 min after the start of the incubation was taken as a measure of the TK activity. The TPP effect was expressed at (TKsat. - TK)/TK x 100. The coefficient of variation for the TPP effect determination was 9.6% within the TPP effect range from 6 to 22%. Within the TPP effect range from 24 to 46%, the coefficient of variation was 8.7%. The mean TPP effect as measured in a group of healthy individuals amounted to 13.5%, with a range from 8.3 to 18.5%. In a group of obese women submitted to an energy-restricted diet, the TPP effect ranged from 12.7 to 30%. In patients suffering from Wernicke's encephalopathy, the TPP effect varied from 28 to 67%.

Changes in some lipid variables in obese women during the early days of fasting.

Adult obese human subjects with a normal or slightly disturbed oral glucose tolerance test, were submitted to a 7-day fast. Initial serum triglyceride levels were inversely related to the Intralipid fractional removal rate, reflecting the close dependency of triglyceridemia on clearance efficiency; the correlation was less significant on the 5th day of fasting. The direction of the change in triglycerides during fasting was clearly related to the prefast triglyceride levels, the groups of patients with a lower level showing an increase, the group with a higher level showing a decrease. Changes in triglyceridemia were inversely related to fractional removal rate; changes in lipid clearing from plasma, however, could not explain the direction of changes in triglyceridemia in all subjects investigated. The group of patients responding to fasting with a decrease in triglyceridemia had a lower mean initial serum cholesterol value and their serum cholesterol levels showed a less prolonged increase during fasting than the other groups of patients.