William P. Fay

Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.

Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 +/- 8 micrograms) than littermate controls (70.5 +/- 8 micrograms, P < 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 +/- 8 micrograms) was not significantly different (P = 0.63) than that of control animals receiving saline (37 +/- 1 micrograms), while hydroxyproline content was significantly increased in heterozygote (77 +/- 12 micrograms, P = 0.06) and wild-type (124 +/- 19 micrograms, P < 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.

Increased Thrombosis After Arterial Injury in Human C-Reactive Protein–Transgenic Mice

Background—C-reactive protein (CRP), an acute-phase reactant long considered merely an innocent bystander in the inflammatory process, is now recognized as a powerful predictor of cardiovascular events. Emerging in vitro evidence suggests that CRP may have direct proinflammatory and prothrombotic effects on monocytes and endothelial cells. To determine whether CRP directly modulates vascular cell function in vivo, we subjected wild-type mice, which do not express CRP, and human CRP–transgenic (CRPtg) mice to 2 models of arterial injury. Methods and Results—Baseline serum CRP levels in CRPtg mice were 18±6 mg/L. CRP levels were undetectable in wild-type mice. Transluminal wire injury led to complete thrombotic occlusion of the femoral artery at 28 days in 75% of CRPtg arteries (6 of 8) compared with 17% (2 of 12) in wild-type mice (P <0.05). In a model of arterial photochemical injury, clot formation time was shortened in CRPtg mice; mean time to occlusion was 33±19 minutes compared with 59±19 minutes in wild-type mice (n=10; P <0.05). Conclusions—Arterial injury in CRPtg mice results in an expedited and higher rate of thrombotic occlusion. This is the first report of a prothrombotic phenotype directly attributable to the presence of human CRP in vivo. Investigation of the inflammatory-thrombotic axis in CRPtg mice may elucidate the prothrombotic actions of CRP in unstable arterial diseases and may pave the way for novel therapeutic interventions for preventing cardiovascular events.

Antidote-mediated control of an anticoagulant aptamer in vivo

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.

Regulation of Arterial Thrombolysis by Plasminogen Activator Inhibitor-1 in Mice

BACKGROUND Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. METHODS AND RESULTS We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). CONCLUSIONS PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.

Plasminogen Activator Inhibitor-1 Is a Major Determinant of Arterial Thrombolysis Resistance

BACKGROUND Platelet-rich thrombi are resistant to lysis by tissue plasminogen activator (tPA). Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of tPA, may contribute to arterial thrombolysis resistance. However, few data are available regarding the effect of PAI-1 on arterial thrombolysis in animals. We used a murine carotid injury model to test the hypothesis that PAI-1 inhibits thrombolysis mediated by pharmacological concentrations of tPA. METHODS AND RESULTS Platelet-rich thrombi were induced in wild-type mice (PAI-1 +/+; n=11) and PAI-1-deficient mice (PAI-1 -/-; n=11) with ferric chloride. Baseline carotid blood flows and mean occlusion times did not differ between PAI-1 +/+ and PAI-1 -/- mice. Clot lysis was induced by infusion of heparin (200 U/kg bolus, 70 U. kg-1. h-1 drip), human plasminogen (50 mg/kg), and tPA at 20 (n=10) or 100 (n=12) microg. kg-1. min-1. Mean plasma tPA antigens were 2.7 microg/mL (tPA infusion, 20 microg. kg-1. min-1) and 5.5 microg/mL (tPA infusion, 100 microg. kg-1. min-1), with no significant differences between PAI-1 +/+ mice and PAI-1 -/- mice. Reperfusion after tPA 20 microg. kg-1. min-1 occurred in 1 of 5 PAI-1 +/+ mice versus 5 of 5 PAI-1 -/- mice (P=0.0006). Reperfusion occurred in all mice that received tPA 100 microg. kg-1. min-1, but reperfusion times were significantly shorter in PAI-1 -/- mice (17. 8+/-2.6 minutes, n=6) than in PAI-1 +/+ mice (35.7+/-5.1 minute, n=6; P=0.01). Histological analyses confirmed that carotid thrombi were platelet rich and that PAI-1 was distributed uniformly throughout thrombi from PAI-1 +/+ mice. Lysates of PAI-1 +/+ platelets inhibited human tPA, whereas PAI-1 -/- platelet lysates did not. CONCLUSIONS PAI-1 is a major determinant of the resistance of platelet-rich arterial thrombi to lysis by pharmacological concentrations of tPA. Strategies to inhibit or resist PAI-1 may enhance thrombolysis.

Chronic Iron Administration Increases Vascular Oxidative Stress and Accelerates Arterial Thrombosis

Background—Iron overload has been implicated in the pathogenesis of ischemic cardiovascular events. However, the effects of iron excess on vascular function and the thrombotic response to vascular injury are not well understood. Methods and Results—We examined the effects of chronic iron dextran administration (15 mg over 6 weeks) on thrombosis, systemic and vascular oxidative stress, and endothelium-dependent vascular reactivity in mice. Thrombus generation after photochemical carotid artery injury was accelerated in iron-loaded mice (mean time to occlusive thrombosis, 20.4±8.5 minutes; n=10) compared with control mice (54.5±35.5 minutes, n=10, P =0.009). Iron loading had no effect on plasma clotting, vessel wall tissue factor activity, or ADP-induced platelet aggregation. Acute administration of dl-cysteine, a reactive oxygen species scavenger, completely abrogated the effects of iron loading on thrombus formation, suggesting that iron accelerated thrombosis through a pro-oxidant mechanism. Iron loading enhanced both systemic and vascular reactive oxygen species production. Endothelium-dependent vasorelaxation was impaired in iron-loaded mice, indicating reduced NO bioavailability. Conclusions—Moderate iron loading markedly accelerates thrombus formation after arterial injury, increases vascular oxidative stress, and impairs vasoreactivity. Iron-induced vascular dysfunction may contribute to the increased incidence of ischemic cardiovascular events that have been associated with chronic iron overload.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life

To investigate the consequence of deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI-deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice produced offspring in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI-deficient mice developed normally, reached adulthood, and were fertile. No gross physical abnormalities were observed up to 24 months of age. Hematological analysis of TAFI-deficient mice did not show any major differences including plasma fibrinogen level, prothrombin time, and activated partial thromboplastin time. TAFI-deficient mice did not suffer from excess bleeding as determined by blood loss following tail transection, although their plasma failed to prolong clot lysis time in vitro. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous injury model. TAFI deficiency did not improve survival rate compared with the wild-type in thrombin-induced thromboembolism, factor X coagulant protein-induced thrombosis, and endotoxin-induced disseminated intravascular coagulation. Furthermore, TAFI deficiency did not alter kaolin-induced writhing response, implying that TAFI does not play a major role in bradykinin catabolism. The current study demonstrates that TAFI deficiency does not change normal responses to acute challenges.

Usefulness of dobutamine stress echocardiography in detecting coronary artery disease in end-stage renal disease

The cardiovascular evaluation of patients with end-stage renal disease (ESRD) has been hampered by the suboptimal sensitivity and specificity of currently employed diagnostic tests. Dobutamine stress echocardiography (DSE) is a recently developed technique which is accurate for the diagnosis of coronary artery disease (CAD) in general populations. The purpose of this study was to assess its diagnostic accuracy and prognostic implications in patients with ESRD. Patients with ESRD (n = 97) underwent DSE as part of a preoperative evaluation before being listed for renal transplantation. Patients were followed for 12 +/- 6 months (range 1 to 24) after the study. Rest and dobutamine stress echocardiograms were analyzed for regional and global function. Coronary angiography was performed in 30 patients, and 25 underwent renal transplantation in the follow-up period. DSE had a sensitivity of 95% (92% for 1-vessel, 100% for > or = 2-vessel disease), specificity of 86%, and accuracy of 90% for the detection of CAD. During the follow-up period, 6 patients died; DSE revealed inducible ischemia in 4, and catheterization before death revealed multivessel CAD in 2. Conversely, a normal DSE identified a very low risk population, with a 97% probability of being free of cardiac complications or death during the follow-up period. We conclude that DSE accurately identifies CAD in patients with ESRD and identifies a cohort of patients at low risk for cardiac complications.

Vascular Functions of the Plasminogen Activation System

The plasminogen activator (PA) system, which controls the formation and activity of plasmin, plays a key role in modulating hemostasis, thrombosis, and several other biological processes. While a great deal is known about the function of the PA system, it remains a focus of intensive investigation, and the list of biological pathways and human diseases that are modulated by normal and pathologic function of its components continues to lengthen. Because of remarkable advances in molecular genetics, the laboratory mouse has become the most useful animal system to study the normal and pathologic functions of the PA system. The purpose of this review is to summarize studies that have used genetically modified mice to examine the functions of the PA system in hemostasis and thrombosis, intimal hyperplasia after vascular injury, and atherosclerosis. Particular emphasis is placed on the vascular functions of PA inhibitor-1, a key regulator of the PA system, and the multiple variables that appear to account for the complex role of PA inhibitor-1 in regulating vascular remodeling. Lastly, the strengths and limitations of using mice to model human vascular disease processes are discussed.

Natural history of hypertrophic cardiomyopathy in the elderly.

The prognosis of patients diagnosed as having hypertrophic cardiomyopathy at advanced age has not been well defined. This study details follow-up information obtained for 95 patients initially diagnosed as having hypertrophic cardiomyopathy at age greater than or equal to 65 years. Seventy-five percent of patients were symptomatic, as defined by the presence of chest pain, dyspnea or syncope, and the mean ventricular septal thickness was 20 mm. The median duration of follow-up study was 4.2 years. The survival rate at 1 and 5 years was 95% and 76%, respectively, which was not significantly different from that an age- and gender-matched control group. Of patients presenting with New York Heart Association functional class I or II dyspnea, only 18% progressed to class III or IV during the follow-up period. However, patients presenting with class III dyspnea had a 1 year mortality rate of 36%, significantly higher than that of control subjects (p less than 0.003). Of the echocardiographic variables, indexed left atrial size was most strongly associated with reduced survival (p less than 0.008). These results suggest that the prognosis of elderly patients with hypertrophic cardiomyopathy is generally favorable. Certain clinical and echocardiographic variables appear to be of use in identifying patients with a less favorable prognosis.