Differential vascular endothelial cell toxicity of established and novel BCR-ABL tyrosine kinase inhibitors.

BCR-ABL tyrosine kinase inhibitors (TKIs) have dramatically improved survival in Philadelphia chromosome-positive leukemias. Newer BCR-ABL TKIs provide superior cancer outcomes but with increased risk of acute arterial thrombosis, which further increases in patients with cardiovascular comorbidities and mitigates survival benefits compared to imatinib. Recent studies implicate endothelial cell (EC) damage in this toxicity by unknown mechanisms with few side-by-side comparisons of multiple TKIs and with no available data on endothelial impact of recently approved TKIs or novels TKIs being tested in clinical trials. To characterize BCR-ABL TKI induced EC dysfunction we exposed primary human umbilical vein ECs in 2D and 3D culture to clinically relevant concentrations of seven BCR-ABL TKIs and quantified their impact on EC scratch-wound healing, viability, inflammation, and permeability mechanisms. Dasatinib, ponatinib, and nilotinib, the TKIs associated with thrombosis in patients, all significantly impaired EC wound healing, survival, and proliferation compared to imatinib, but only dasatinib and ponatinib impaired cell migration and only nilotinib enhanced EC necrosis. Dasatinib and ponatinib increased leukocyte adhesion to ECs with upregulation of adhesion molecule expression in ECs (ICAM1, VCAM1, and P-selectin) and leukocytes (PSGL1). Dasatinib increased permeability and impaired cell junctional integrity in human engineered microvessels, consistent with its unique association with pleural effusions. Of the new agents, bafetinib decreased EC viability and increased microvessel permeability while asciminib and radotinib did not impact any EC function tested. In summary, the vasculotoxic TKIs (dasatinib, ponatinib, nilotinib) cause EC toxicity but with mechanistic differences, supporting the potential need for drug-specific vasculoprotective strategies. Asciminib and radotinib do not induce EC toxicity at clinically relevant concentrations suggesting a better safety profile.

A tissue-engineered model of the blood-tumor barrier during metastatic breast cancer.

Metastatic brain cancer has poor prognosis due to challenges in both detection and treatment. One contributor to poor prognosis is the blood-brain barrier (BBB), which severely limits the transport of therapeutic agents to intracranial tumors. During the development of brain metastases from primary breast cancer, the BBB is modified and is termed the 'blood-tumor barrier' (BTB). A better understanding of the differences between the BBB and BTB across cancer types and stages may assist in identifying new therapeutic targets. Here, we utilize a tissue-engineered microvessel model with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs) and surrounded by human breast metastatic cancer spheroids with brain tropism. We directly compare BBB and BTB in vitro microvessels to unravel both physical and chemical interactions occurring during perivascular cancer growth. We determine the dynamics of vascular co-option by cancer cells, modes of vascular degeneration, and quantify the endothelial barrier to antibody transport. Additionally, using bulk RNA sequencing, ELISA of microvessel perfusates, and related functional assays, we probe early brain endothelial changes in the presence of cancer cells. We find that immune cell adhesion and endothelial turnover are elevated within the metastatic BTB, and that macrophages exert a unique influence on BTB identity. Our model provides a novel three-dimensional system to study mechanisms of cancer-vascular-immune interactions and drug delivery occurring within the BTB.

Development of a Colic Scoring System to Predict Outcome in Horses.

Acute abdominal pain in the horse is a common emergency presenting to equine practices. The wide variety of etiologies makes prognosticating survival a challenge. A retrospective, multi-institutional clinical study was performed to determine clinical parameters associated with survival of horses with colic, and to use them to develop a colic survival scoring system. The scoring system was then validated using clinical data in the prospective portion of the study. Medical records from 67 horses presenting for acute abdominal pain were evaluated to develop the colic assessment score. Twenty eight variables were compared between survivors and non-survivors and entered into logistic regression models for survival. Of these, six variables were included in the colic assessment score. A total colic assessment score range was from 0 to 12, with the highest score representing the lowest probability of survival. The optimal cutoff value to predict survival was seven resulting in an 86% sensitivity and 64% specificity with a positive predictive value of 88% and a negative predictive value of 57%. Data from 95 horses presenting for abdominal pain to two equine hospitals was then collected prospectively to validate the colic assessment score. Horses from the prospective portion of the study that received a score >7 were classified as predicted to die and those with a score ≤7 were predicted to survive. The classification was compared to the actual outcome, of which the sensitivity, specificity, positive and negative predictive values of the colic assessment score were 84, 62, 88, and 52%, respectively.

Tissue-engineered blood-brain barrier models via directed differentiation of human induced pluripotent stem cells.

Three-dimensional (3D) tissue-engineered models of the blood-brain barrier (BBB) recapitulate in vivo shear stress, cylindrical geometry, and cell-ECM interactions. Here we address four issues associated with BBB models: cell source, barrier function, cryopreservation, and matrix stiffness. We reproduce a directed differentiation of brain microvascular endothelial cells (dhBMECs) from two fluorescently labeled human induced pluripotent stem cell lines (hiPSCs) and demonstrate physiological permeability of Lucifer yellow over six days. Microvessels formed from cryopreserved dhBMECs show expression of BBB markers and maintain physiological barrier function comparable to non-cryopreserved cells. Microvessels displaying physiological barrier function are formed in collagen I hydrogels with stiffness matching that of human brain. The dilation response of microvessels was linear with increasing transmural pressure and was dependent on matrix stiffness. Together these results advance capabilities for tissue-engineered BBB models.

Human iPSC-derived blood-brain barrier microvessels: validation of barrier function and endothelial cell behavior.

Microvessels of the blood-brain barrier (BBB) regulate transport into the brain. The highly specialized brain microvascular endothelial cells, a major component of the BBB, express tight junctions and efflux transporters which regulate paracellular and transcellular permeability. However, most existing models of BBB microvessels fail to exhibit physiological barrier function. Here, using (iPSC)-derived human brain microvascular endothelial cells (dhBMECs) within templated type I collagen channels we mimic the cylindrical geometry, cell-extracellular matrix interactions, and shear flow typical of human brain post-capillary venules. We characterize the structure and barrier function in comparison to non-brain-specific microvessels, and show that dhBMEC microvessels recapitulate physiologically low solute permeability and quiescent endothelial cell behavior. Transcellular permeability is increased two-fold using a clinically relevant dose of a p-glycoprotein inhibitor tariquidar, while paracellular permeability is increased using a bolus dose of hyperosmolar agent mannitol. Lastly, we show that our human BBB microvessels are responsive to inflammatory cytokines via upregulation of surface adhesion molecules and increased leukocyte adhesion, but no changes in permeability. Human iPSC-derived blood-brain barrier microvessels support quantitative analysis of barrier function and endothelial cell dynamics in quiescence and in response to biologically- and clinically-relevant perturbations.

Enaminones 12. An explanation of anticonvulsant activity and toxicity per Linus Pauling's clathrate hypothesis.

The x-ray crystal structure of 3-((5-methylisoxazol-3-yl)amino)-5-methylcyclohex-2-enone (12b) and 3-((5-methylisoxazolyl-3-yl)amino)-5,5-dimethylcyclohex-2-enone (12c) were determined and correlated to their anticonvulsant activity in mice and rats. A hypothesis for the toxicity of the analogs are advanced. In addition, a series of 5-methyl-N-(3-oxocyclohex-1-enyl)-isoxazole-3-carboxamides were synthesized and evaluated for anticonvulsant activity. These compounds were compared to the activity of the corresponding amino and aminomethyl enaminones. Additional investigation involved the synthesis and evaluation of a trifluoromethyl analog of the active isoxazole tert-butyl 4-(5-methisoxazol-3-yl-amino)-6-methyl-2-oxo-cyclohex-3-ene carboxylate (4f).