Ronald Konopka

PULMONARY VASCULAR REMODELING DISTAL TO PULMONARY ARTERY LIGATION IS ACCOMPANIED BY UPREGULATION OF ENDOTHELIN RECEPTORS AND NITRIC OXIDE SYNTHASE

There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8 +/- 2.2 microns vs. 16.8 +/- 0.9 microns in sham group; P < .005) and number of bronchial arteries per bronchiole (3.21 +/- mu 0.26 vs. 1.86 +/- mu 0.21 in sham group; P < .001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These findings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1-mediated vascular remodeling.There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8+/-2.2 mum vs.16.8+/-0.9 mum in sham group; P <. 005) and number of bronchial arteries per bronchiole (3.21+/-mu0.26vs.1.86+/-mu0.21 in sham group; P <. 001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These ndings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1 mediated vascular remodeling.

Chronic pulmonary thromboembolism in dogs treated with tranexamic acid.

BackgroundMany questions remain regarding the pathogenesis, natural history, diagnosis, and treatment of chronic thromboembolic pulmonary hypertension in patients. To answer such questions, we developed an animal model of this disorder. The brisk thrombolytic response of canines to acute embolism has, previously, prevented the establishment of such a model. Methods and ResultsThe fibrinolytic inhibitor tranexamic acid was given orally to canines before, and for intervals after, pulmonary emboli were released from venous thrombi formed in vivo in femoral veins or the inferior vena cava. Preliminary studies disclosed that embolic residuals from femoral vein thrombi were not sufficient to cause significant, persistent pulmonary hypertension. With repetitive, larger thrombi embolized from the inferior vena cava, however, persistent pulmonary hypertension was achieved in most animals. ConclusionsResolution of emboli in the canine can be inhibited by tranexamic acid. As in humans, a spectrum of embolic residuals is encountered, and the perfusion lung scan consistently underestimates the extent of embolic residuals. Studies of this animal model continue.

Factors contributing to increased vascular fibrinolytic activity in mongrel dogs.

BackgroundNumerous investigators have observed that pulmonary emboli are rapidly lysed in a canine model system. This study was undertaken to delineate the unique mechanism that accounts for the rapid dissolution of pulmonary emboli in mongrel dogs Methods and ResultsCanine plasminogen activator (PA) activity (2.6±1.1 IU/mL acidified platelet-poor plasma [PPP], <0.3 IU/mL acidified whole blood serum [WBSJ, mean±SD; n=6) and PA inhibitor activity (6.1±2.6 U/mL PPP, 35.4±7.8 U/mL WBS; n=6) were determined in standard plasminogen-based chromogenic assays. Analysis of canine PPP, WBS, platelet lysates, and primary canine endothelial cell (EC) cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography revealed a plasminogen-dependent lytic zone at 45-kd relative molecular mass that was shown to be related to urokinase-type PA (u-PA) by its selective inhibition through amiloride. Analysis of canine platelets on standard 125I fibrin plate assays revealed a net fibrinolytic activity. In a clot lysis assay system, canine platelets were able to stimulate fibrinolysis when layered on the outside of fibrin clots containing autologous PPP. Moreover, net fibrinolytic activity of primary canine pulmonary artery endothelial cells was higher than the activities expressed by canine aortic or carotid artery endothelial cells. ConclusionRapid lysis of pulmonary emboli in mongrel dogs appears to be a result of 1) the high u-PA activity in canine PPP and 2) the predominant association of u-PA activity with canine platelets and canine pulmonary artery endothelial cells. (Circulation 1993;87:1990-2000)

Suppression of thrombolysis in a canine model of pulmonary embolism.

BackgroundThe brisk fibrinolytic response of canines has impaired efforts to develop a canine model of chronic thromboembolic pulmonary hypertension. Difficulties in retaining chronic embolic residuals were partially overcome by administration of tranexamic acid (TXA) (Circulation. 1991;83:1272–1279.). In this study, we used type 1 plasminogen activator inhibitor (PAI-1), a major inhibitor of the endogenous fibrinolytic system, to determine its efficacy in the suppression of thrombolysis in canines. Methods and ResultsThrombus was induced in the inferior vena cava of anesthetized mongrel dogs with thrombin and a special double-balloon catheter; 2 hours later, the thrombus was embolized. In one group of dogs, activated type 1 plasminogen activator inhibitor (PAI-1) (130 μg) was delivered directly into the forming thrombus; in another, TXA (110 mg/kg) was given intravenously before thrombus formation; in controls, thrombus was induced without inhibitors. Cross-linked fibrin degradation product (D-dimer) appeared in the blood of control animals within 1 hour of thrombus induction (176 ± 62.5 versus 1.02 ± 0.39 ng/mL baseline; mean ± SEM), was maximal by 4 hours (413 ± 110 ng/mL) and remained elevated at 24 hours (90.8 ± 19.5 ng/mL). Compared with controls, PAI- 1 and TXA suppressed D-dimer release by 80% and 85%, respectively, over the first 24 hours. One week later, animals were killed, and residual emboli were harvested. Perfusion scan defects persisted in all animals at this time, but there were no scan defect differences among groups. However, emboli recovered from animals receiving PAI-1 still harbored immunoreactive PAI-1 and were, on average, more than twofold greater in mass (393 ± 56 mg) than emboli recovered from either controls (183 ± 76 mg) or animals receiving TXA (180 ± 80 mg). ConclusionsIntravenous TXA and intrathrombus PAI-1 effectively suppress thrombolysis for 24 hours in canines. Thromboemboli enriched with PAI-1 appear to resist lysis for longer periods of time (up to 1 week). These findings are consistent with the hypothesis that PAI-1 remains associated with the embolus, where it continues to inhibit lysis, whereas TXA eventually diffuses out of the embolus, allowing lysis to ensue.

Indium-111-labeled platelets: effects of heparin on uptake by venous thrombi and relationship to the activated partial thromboplastin time.

The goal of heparin therapy in deep vein thrombosis is to prevent thrombus extension. The relationship between thrombus extension and the results of coagulation tests used to monitor heparin therapy is unclear. To explore this relationship, we studied the effect of several heparin regimens on the accretion of indium-111-labeled platelets on fresh venous thrombi, as detected by gamma imaging, and monitored the activated partial thromboplastin time (APTT). Six dogs were treated with a 300-U/kg bolus of heparin followed by a 90-U/kg/hour heparin infusion, a dose of heparin sufficient to increase the APTT to levels greater than eight times baseline (APTT ratio); platelet accretion (thrombus imaging) occurred only after the heparin effect was reversed with protamine sulfate. Nineteen dogs were treated with a 150-U/kg bolus of heparin followed by a 4-hour, 45-U/kg/hour heparin infusion; a thrombus was demonstrated only after protamine injection in 12 (mean APTT ratio 1.3 0.19) and before protamine injection in seven. In thirteen of these 19 dogs, 30 minutes separated the platelet injection from heparin therapy, while in six this duration was less than 30 minutes. In four of these six dogs, thrombi were demonstrated before protamine therapy and at APTT ratios greater than 3.0. Finally, 10 dogs were treated with a 100-UIkg bolus followed by a 3-hour, 50-U/kg/hour heparin infusion, after which the APTT was allowed to return to baseline values spontaneously. In all 10 dogs, a thrombus was demonstrated only after cessation of the heparin infusion, and at a mean APTT ratio of 1.4 ± 0.15 times baseline. These results suggest that, except with very early platelet injection, platelet accretion by thrombi is consistently inhibited by heparin at APTT ratios greater than 2.5. Platelet accretion by venous thrombi occurs within narrow limits of heparin effect as reflected by the APTT.

Improved imaging of deep venous thrombi during anticoagulation using radiolabelled anti-D-dimer antibodies.

ObjectivesRadiolabelled anti-fibrin antibodies have not yet enabled reliable and practical diagnosis of venous thromboembolism. However, previous unsuccessful clinical trials were performed with anti-fibrin &bgr;-chain antibodies that do not optimally bind to thrombi during anticoagulation. The current experiments were performed to determine if radiolabelled anti-D-dimer antibodies more reliably allowed nuclear medicine imaging of deep venous thrombi during anticoagulation than anti-&bgr;-chain antibodies. MethodsDogs with pre-existing unilateral femoral vein thrombi were anticoagulated with heparin (300 units·kg−1 bolus followed by 90 units·kg−1·h−1 continuous infusion). They were then allocated to receive one of three 111In labelled antibodies: anti-D-dimer, anti-&bgr; or a non-specific control antibody. Gamma scans of the legs were performed at regular intervals for 24 h. Scans were interpreted in a blinded fashion and the number of gamma counts from the femoral area on the thrombosed side was compared to the contralateral side. Clot/blood isotope density ratios were determined post-mortem. ResultsLeg thrombi were 100% detectable in the anti-D-dimer group, but only 60% detectable in the anti-&bgr; group. Mean±SD relative counts in the thrombosed femoral area was 137±10% compared to the contralateral side in the anti-D-dimer group, but only 116±20% in the anti-&bgr; group. The clot/blood ratio was 24.5±2.8 in the anti-D-dimer group, but only 7.8±2.0 in the anti-&bgr; group. Conclusions111In labelled anti-D-dimer provides superior nuclear medicine images of thrombi during intensive anticoagulation compared to anti-&bgr;. Clinical results with radiolabelled anti-D-dimer may be more promising than those previously observed with other anti-fibrin antibodies.

Hemodynamics and Gas Exchange During Angioscopy in the Dog

We have previously developed a technique for the in vivo visualization of the pulmonary arteries in the experimental animal with a fiberoptic instrument (angio scope). To assess the potential hemodynamic and gas exchange effects of angioscopy, we studied five dogs before and after pulmonary embolization. Sequential observations were made of arterial blood gases, mean arterial pressure, pulmonary artery pressure, heart rate, cardiac output, and the electrocardiogram. The most common arrhythmias were ventricular premature contractions which were comparable to those seen with right heart catheterization. Statistically significant, but clinically minor, effects were found on cardiac output, mean arterial pressure, and heart rate in dogs when angioscopy was performed after pulmonary embolization. We conclude that fiberoptic angioscopy does not induce deleterious effects on hemodynamics or gas exchange in the experimen tal animal, prior to or after embolization.

Magnetic resonance detection of acute pulmonary emboli in a canine model with pathologic correlation

RATIONALE AND OBJECTIVES The authors evaluated the accuracy of magnetic resonance (MR) imaging in depicting acute pulmonary emboli at the lobar, segmental, and subsegmental levels. METHODS The authors induced 29 autologous emboli in five dogs and confirmed their location with angiography and anatomic dissection. MR images obtained with four sequences were independently evaluated by two radiologists to detect emboli in each vascular segment. Sensitivities, specificities, and accuracies were calculated at segmental and lobar levels. RESULTS The fast short-tau inversion-recovery images provided the greatest conspicuity and highest overall accuracy (reader 1 = 74.3%, reader 2 = 80%). Accuracy of two-dimensional fast multiplanar spoiled gradient-recalled-echo images was limited by spatial resolution (reader 1 = 71.4%, reader 2 = 74.3%). The fast spin-echo T2-weighted and spin-echo T1-weighted sequences were intermediate in their depiction of acute emboli. Similar results were seen at the lobar level. CONCLUSION MR images depict acute pulmonary embolism at the segmental and lobar levels with reasonable accuracy. Fast short-tau inversion-recovery sequences provided the greatest sensitivity and accuracy.

Inhibition of indium-111 platelet accretion onto venous thrombi in dogs by prostacyclin.

The known platelet anti-aggregant effects of prostacyclin (epoprostenol) suggest that it may have therapeutic potential in conditions in which the platelet plays a pathophysiological role. The growth of venous thrombi is one such condition. We have attempted to determine, in a canine model of fresh venous thrombosis, whether prostacyclin infusion inhibits platelet accretion in vivo and how this in vivo event related to hemodynamic and in vitro platelet anti-aggregant effects. Gamma camera imaging over thrombi for accretion of indium-111-labeled platelets disclosed that prostacyclin, at an infusion rate of 50 ng/kg per min, inhibited platelet accretion in vivo and resulted in a 95 +/- 4% decrease in in vitro adenosine diphosphate-induced platelet aggregation, and a decrease in mean arterial pressure to 86 +/- 4% of pre-infusion values. Step-wise decrements of prostacyclin infusion demonstrated that platelet accretion occurred in vivo at infusion rates of approximately 10-20 ng/kg per min and correlated with an in vitro adenosine diphosphate-induced aggregation of 54 +/- 13% of control values. Thus, prostacyclin, in a dose that causes only a mild decrease in systemic pressure, can completely inhibit platelet uptake onto fresh venous thrombi in the dog, and this inhibition correlates closely with in vitro adenosine diphosphate-induced platelet aggregation. The potential therapeutic implications of these findings are discussed.