Background– Heparin anticoagulation has been used successfully for cardiopulmonary bypass (CPB). However, an alternative anticoagulant approach is desirable due to the cases of heparin-induce thrombocytopenia. Dabigatran provides anticoagulation for an in-vitro model of simulated CPB. The current analysis tests the hypothesis that dabigatran provides sufficient anticoagulation for CPB in intact rabbits. Methods – Nonlinear mixed effects models were used to estimate dabigatran parameters for a 2-compartment pharmacokinetic model in 10 New Zealand White rabbits. A dabigatran infusion designed to maintain a plasma concentration of 90 µg/mL was run throughout CPB based on the pharmacokinetics. Animals were subjected to sternotomy, and anticoagulated with intravenous dabigatran (6 animals) or heparin (4 animals). Rabbits were cannulated centrally using the right atrium and ascending aorta and CPB was maintained for 120 minutes. Measurement of activated clotting time, thromboelastometric reaction time (R), and blood gases were performed during CPB. Then, the animals were sacrificed and the brain and one kidney were removed for histology. Sections of the arterial filters were inspected using electron microscopy. Results – The observed dabigatran concentrations during CPB were above the target concentration, ranging from 137 ± 40 μg/mL at 5 min of CPB to 428 ± 150 μg/mL at 60 min, and 295 ± 35 μg/mL at 120 min. All rabbits completed 2 hours of CPB without visible thrombosis. In the two groups reaction time (R) values were elevated, reaching 10262 ± 4198 sec (dabigatran group) and 354 ± 141 sec (heparin group) at 120 min of CPB. Brains and kidneys showed no evidence of thrombosis or ultrastructural damage. Sections of the arterial line filter showed minimal or no fibrin. There was no significant difference in outcomes between dabigatran and heparin treated animals. Conclusions – In this first-use, proof of concept study, we have shown that dabigatran provides acceptable anticoagulation similar to heparin to prevent thrombosis using a rabbit CPB model.

Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease caused by smooth muscle cell-like tumors containing tuberous sclerosis (TSC) gene mutations and found almost exclusively in females. Patient studies suggest LAM progression is estrogen-dependent, an observation supported by in vivo mouse models. However, in vitro data using TSC-null cells lines demonstrate modest estradiol responses, suggesting estradiol effects in vivo may involve pathways independent of direct tumor stimulation. We previously reported tumor-dependent neutrophil expansion and promotion of TSC2-null tumor growth in an estradiol-sensitive LAM mouse model. We therefore hypothesized that estradiol stimulates tumor growth in part by promoting neutrophil production. Here we report that estradiol-enhanced lung colonization of TSC2-null cells is indeed dependent on neutrophils. We demonstrate that estradiol induces granulopoiesis via ERα in male and female bone marrow cultures. With our novel TSC2-null mouse myometrial cell line, we show that factors released from these cells drive estradiol-sensitive neutrophil production. Lastly, we analyzed single-cell RNA sequencing data from LAM patients and demonstrate the presence of tumor-activated neutrophils. Our data suggest a powerful positive feedback loop whereby estradiol and tumor factors induce neutrophil expansion, which in turn intensifies tumor growth and production of neutrophil-stimulating factors, resulting in continued TSC2-null tumor growth.

Introduction:Dabigatran is an anticoagulant with potential use during cardiopulmonary bypass in children and adults. The pharmacokinetic-pharmacodynamic relationship for dabigatran anticoagulation effect was investigated in an intact animal model using rabbits.

Lymphangioleiomyomatosis (LAM) is a rare lung disease seen almost exclusively in female sexed individuals and characterized by slowly growing, metastatic smooth muscle cell-like adenomas that cause cyst formation in the lung parenchyma and irreversible loss of pulmonary function. Given the female specificity of disease manifestation, metastatic nature, and estrogen sensitivity of LAM cells, we previously proposed that LAM cells originate in the myometrium. Our subsequent reports showed that inactivation of TSC2 in the mouse uterus results in notable LAM features in the setting of primary myometrial tumors. Additionally, we established that estrogen is required to maintain LAM-like tumor progression in a uterine-specific Tsc2-knockout murine model. However, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol in vitro, suggesting that estradiol may act elsewhere in vivo to promote LAM progression. Recent immunophenotyping of LAM-like TSC2-null uterine tumors revealed preferential increase in polymorphonuclear cell (PMNs) numbers of the tumor microenvironment; we also determined that PMNs are important regulators of tumor growth. Therefore, we hypothesize that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Here, we show that estrogen availability influences PMN mobilization in naïve and tumor-burdened mice. Using bone marrow cultures and CFC assay, we demonstrate that estradiol is a potent inducer of PMN production in chronic inflammatory conditions. Employing both pharmacologic agents and estrogen receptor (ER)-null bone marrow, we showed that ERα is necessary for promoting induction of PMN fate for myeloid progenitors. While we see that the migratory, invasive, and proliferative capacities of TSC2-null cell lines are not augmented when stimulated with E2 directly, experiments are underway to assess the extent to which estrogen promotes the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. Together these data provide insight into the robust effect of in vivo estrogen stimulation as estradiol may be a dual effector in LAM tumor progression and great target for anti-LAM therapeutic strategy. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.