Amy Lankford

Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.

Pertussis toxin sensitive and insensitive effects of adenosine and carbachol in murine atria overexpressing A1-adenosine receptors

It was investigated how A1‐adenosine receptor overexpression alters the effects of carbachol on force of contraction and beating rate in isolated murine atria. Moreover, the influence of pertussis toxin on the inotropic and chronotropic effects of adenosine and carbachol in A1‐adenosine receptor overexpressing atria was studied. Adenosine and carbachol alone exerted negative inotropic and chronotropic effects in electrically driven left atrium or spontaneously beating right atrium of wild‐type mice. These effects were abolished or reversed by pre‐treatment of animals with pertussis toxin which can interfere with signal transduction through G‐proteins. Adenosine and carbachol exerted positive inotropic but negative chronotropic effects in atrium overexpressing A1‐adenosine receptors from transgenic mice. The positive inotropic effects of adenosine and carbachol were qualitatively unaltered whereas the negative chronotropic effects were abolished or reversed in atrium overexpressing A1‐adenosine receptors after pre‐treatment by pertussis toxin. Qualitatively similar effects for adenosine and carbachol were noted in the presence of isoprenaline, β‐adrenoceptor agonist. It is concluded that overexpression of A1‐adenosine receptors also affects the signal transduction of other heptahelical, G‐protein coupled receptors like the M‐cholinoceptor in the heart. The chronotropic but not the inotropic effects of adenosine and carbachol in transgenic atrium were mediated via pertussis toxin sensitive G‐proteins.

Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression

OBJECTIVES To identify potential molecular genetic determinants of cardiovascular ischemic tolerance in wild-type and transgenic hearts overexpressing A(1) adenosine receptors (A(1)ARs). METHODS cDNA microarrays were used to explore expression of 1824 genes in wild-type hearts and ischemia-tolerant mouse hearts overexpressing A(1)ARs. RESULTS Overexpression of A(1)ARs reduced post-ischemic contractile dysfunction, limited arrhythmogenesis, and reduced necrosis by approximately 80% in hearts subjected to 30 min global ischemia 60 min reperfusion. Cardioprotection was abrogated by acute A(1)AR antagonism, and only a small number (19) of genes were modified by A(1)AR overexpression in normoxic hearts. Ischemia-reperfusion significantly altered expression of 75 genes in wild-type hearts (14 induced, 61 down-regulated), including genes for metabolic enzymes, structural/motility proteins, cell signaling proteins, defense/growth proteins, and regulators of transcription and translation. A(1)AR overexpression reversed the majority of gene down-regulation whereas gene induction was generally unaltered. Additionally, genes involved in cell defence, signaling and gene expression were selectively modified by ischemia in transgenic hearts (33 induced, 10 down-regulated), possibly contributing to the protected phenotype. Real-time PCR verified changes in nine selected genes, revealing concordance with array data. Transcription of the A(1)AR gene was also modestly reduced post-ischemia, consistent with impaired functional sensitivity to A(1)AR stimulation CONCLUSIONS Data are presented regarding the early post-ischemic gene profile of intact heart. Reduced A(1)AR transcription is observed which may contribute to poor outcome from ischemia. A(1)AR overexpression selectively modifies post-ischemic gene expression, potentially contributing to ischemic-tolerance.

A1 adenosine receptor overexpression decreases stunning from anoxia-reoxygenation: role of the mitochondrial KATP channel

Abstract Myocardial A1 adenosine receptor (A1AR) overexpression protects hearts from ischemia-reperfusion injury; however, the effects during anoxia are unknown. We evaluated responses to anoxia-reoxygenation in wild-type (WT) and transgenic (Trans) hearts with ∼200-fold overexpression of A1ARs. Langendorff perfused hearts underwent 20 min anoxia followed by 30 min reoxygenation. In WT hearts peak diastolic contracture during anoxia was 45 ± 3 mmHg, diastolic pressure remained elevated at 18 ± 3 mmHg after reoxygenation, and developed pressure recovered to 52 ± 4 % of pre-anoxia. A1AR overexpression reduced hypoxic contracture to 29 ± 4 mmHg, and improved recovery of diastolic pressure to 8 ± 1 mmHg and developed pressure to 76 ± 3 % of pre-anoxia. Mitochondrial KATP blockade with 100 μM 5-hydroxydecanoate (5-HD) increased hypoxic contracture to 73 ± 6 mmHg in WT hearts, reduced post-hypoxic recoveries of both diastolic (40 ± 5 mmHg) and developed pressures (33 ± 3 %). In contrast, 5-HD had no effect on hypoxic contracture (24 ± 8 mmHg), or post-hypoxic diastolic (10 ± 2 mmHg) and developed pressures (74 ± 3 %) in Trans hearts. In summary, (i) A1AR overexpression improves myocardial tolerance to anoxia-reoxygenation, (ii) intrinsic mitochondrial KATP channel activation decreases hypoxic contracture and improves functional recovery in wild-type hearts, and (iii) mitochondrial KATP channels do not appear to play a major role in the functional protection from anoxia afforded by A1AR overexpression.

Abstract CT220: A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic canc

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Immunotherapy has recently emerged as a promising modality in cancer treatment, but little is known about the application of this modality in pancreatic cancer (PC). Tumor-infiltrating lymphocytes (TILs) play a major role in anti-tumor immune responses, and their presence is correlated with survival in a variety of tumors. These TILs do not reach the PC cells in significant numbers due to the presence of stroma and a suppressive microenvironment. One of the leading causes for immune suppression is elevated expression of PD-L1 either by the tumor cells or the surrounding regulatory cells, resulting in dysfunction of TILs. Neoadjuvant chemoradiation therapy (CRT) has been advocated as a potential way to improve outcomes of patients with resectable or borderline resectable PC. More importantly, there is recent evidence to suggest that CRT can increase the presence of TILs in the PC microenvironment (PCME), leading to production of interferon-γ (IFN-γ), which could increase the expression of PD-L1 through a negative feedback loop. Accordingly, we hypothesize that blocking the PD-1 receptor will synergize with CRT to increase the density and activation of TILs in the PCME. Methods: This is a prospective multicenter randomized trial which will accrue subjects with resectable or borderline resectable pancreatic cancer who had not received prior treatment for PC. The primary objectives of the study are: (1) to determine the safety of neoadjuvant CRT in combination with Pembrolizumab. (2) To estimate the difference in the number of TILs in pancreatic cancer subjects receiving neoadjuvant CRT in combination with Pembrolizumab to the number of TILs in subjects receiving neoadjuvant CRT alone. This study will also investigate the effect of CRT+/-anti-PD-1 on the other effector and suppressive immune cells and immune checkpoints in PCME. Eligible subjects will be randomized 2:1 to the investigational treatment (Arm A) to receive Pembrolizumab administered IV every 3 weeks on days 1, 22, and 43 during concurrent CRT with capecitabine (825 mg/m2 orally twice daily, Monday through Friday, on days of radiation only) and radiation (50.4 Gy in 28 fractions over 28 days) or Arm B to receive only concurrent CRT with capecitabine. In all subjects, restaging CT scan or MRI will be performed at 4-6 weeks after completion of neoadjuvant treatment to determine resectability. Patients without local or distant disease progression will be taken to the operative room for planned surgery (within 2 weeks of imaging). Postoperatively, resected patients will receive off study standard of care adjuvant gemcitabine (1000mg/kg IV weekly for 3 out of 4 weeks for 6 months). Post operatively resected patients will be followed for up for PFS and OS for up to 2 years. Citation Format: Matthew H.G Katz, Todd W. Bauer, Gauri Rajani Varadhachary, Reid B. Adams, Amy R. Lankford, Gina Petroni, Timothy N. Bullock, Craig L. Slingluff, Osama E. Rahma. A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic canc [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT220. doi:10.1158/1538-7445.AM2015-CT220