Gamb, Scott I.

Background Extracellular DNA traps (ETs) have important implications in both thrombosis and thrombolysis. Thus, developing benchtop thrombus analogs that recapitulate clinical ETs is potentially of great value for preclinical development and testing of thrombolytic agents and thrombectomy devices. In this study, we aimed to develop ETs-rich thrombus analogs for preclinical testing. Methods Red blood cell (RBC)-rich, fibrin-rich, and platelet-rich thrombus analogs were created using human whole blood, platelet-poor plasma, and platelet-rich plasma obtained from the blood bank following institutional approval. Peripheral blood mononuclear cells (9.9×106 cells/mL) isolated from human whole blood and lipopolysaccharide (1 µg/mL) were added to induce ETs. Histochemical, immunohistochemistry and immunofluorescence were used to identify thrombus components and ETs. Scanning electronic microscopy was used to investigate the ultrastructure of the thrombus analogs. The thrombus compositions, morphologic features of ETs and citrullinated histone H3 (H3Cit) expression were compared with those of thrombi retrieved from patients by thrombectomy. Results ETs-rich thrombus analogs were more compacted th-an the ETs-poor thrombus analogs. ETs were identified in both ETs-rich thrombus analogs and patient thrombi showing morphologic features including nuclear lobulation, nuclear swelling, diffused chromatin within cytoplasm, DNA/chromatin extending intracellularly and extracellularly, and extracellular chromatin patches and bundles. In the ETs-poor thrombus analogs, ETs were not observed and H3Cit expression was absent to minimal. The compositions and H3Cit expression in the ETs-rich thrombus analogs fell in the range of patient thrombi. Conclusions ETs-rich thrombus analogs can be consistently created in vitro and may benefit the preclinical development and testing of new thrombolytic agents and thrombectomy devices.

Abstract Background and aims Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard‐of‐care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin‐dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive. Approach and results Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy‐induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine. Conclusions As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

Abstract Background Platelet dense granule (DG) deficiencies, including Hermansky-Pudlak syndrome, are relatively common and frequently under-diagnosed hereditary platelet hypofunctional disorders. Among various laboratory tests, platelet whole mount (WM) transmission electron microscopy (TEM) is considered the gold standard method for diagnosing DG deficiencies. Nevertheless, platelet DG TEM testing is still largely a research tool and has not been thoroughly validated and standardized as a clinical test. Furthermore, reference ranges (RR) of DG count/platelet (DGC) have not been adequately established. Aim Our goals were to validate and standardize platelet WM TEM testing and establish reference ranges for platelet DGC. Methods Based on previously established methods (Blood, 33:598-606), we first optimized and standardized various pre-analytical, analytical and post-analytical procedures including sample matrix, sample stability, platelet-rich plasma (PRP) preparation, platelet mounting procedure and DG counting criteria. Then whole blood samples in ACD-B tubes were collected from a total of 128 healthy donors (64 males and 64 females). Platelet DGC, percentage of platelets without any DG (empty platelets), platelet count (PC), mean platelet volume (MPV) and immature platelet fraction (IPF) were measured. In addition, platelet aggregometry analyses were performed on citrated PRP samples from the same donors. Standard statistical analyses were employed in this study. Results DGC and platelet ultra-structure were stable when whole blood samples were stored at room temperature (RT) for up to 4 days. Reproducible DGC and adequate preservation of platelet ultrastructure depended on an optimized mounting method. The DG counting criteria were developed based on previous publications and guidance from Dr. James G. White. Using the same WM TEM images and DG counting criteria, agreements among different technologists (n=5) improved from 60% to 95%. Finally, DGC reference range study using 128 healthy donor samples (age range 18, 72 years) and scoring 100-200 platelets from each donor showed that the DGC distribution of each donor sample was left-skewed with a median of 15% of empty platelets (range: 3, 40). The median DGC was 2.6 with a range of 1.0—5.1. Reference ranges established at the lower 95th percentile for DGC empty platelets is ≤ 31% (95% CI 28, 34) and for the mid-95th percentiles of DGCs between 1.5 and 4.0. DGCs were weakly associated with MPV and IPF (ρ between 0.22 and 0.28); and inversely associated with the percentage of empty platelets (ρ between -0.26 and -0.25). DGC showed no significant association with PC, platelet maximum aggregation or primary slope of aggregation, nor age or gender. Conclusion We validated platelet WM TEM procedures and for the first time established an adult reference range for platelet DGC. The results of association analyses implied that immature platelets may have slightly more DGs. Disclosures: No relevant conflicts of interest to declare.

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/role-of-foxo3a-in-heart-failure/ .