Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multi-organ dysfunction that is especially severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improve clinical outcomes. To better understand these clinical issues, we performed the first mRNA-sequencing on total circulating leukocytes from neonatal patients undergoing CPB. Our data identified myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, interleukin-8 (IL8) and tumor necrosis factor-α (TNFα) were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate the molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions including artificial surfaces, high shear stress, and cooling/rewarming. Shear stress was found to drive cytokine upregulation via calciumdependent signaling pathways. We also observed that a subpopulation of THP-1 cells died via TNFα-mediated necroptosis, which we hypothesize contributes to post-CPB inflammation. Together, our study identifies a shear-stress modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to demonstrate that shear-stress causes necroptosis. Finally, we observe that calcium and TNFα signaling are novel targets to ameliorate post-CPB inflammation.

Objectives To investigated the effect of Granulocyte-colony stimulating factor (G-CSF) on expression of proteins that regulate apoptosis in newborn piglet brain following cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). Methods The newborn piglets were assigned to three groups: 1/DHCA (30 min of DHCA, 1hr of low-flow CPB), 2/DHCA with prior injection of G-CSF (17µg/kg 2hrs before CPB) and 3/sham-operated. After 2hrs of post-bypass recovery the frontal cortex, striatum and hippocampus were dissected. The expression of proteins was measured by gel electrophoresis or protein arrays. Data are presented in arbitrary units. Statistical analysis was performed using one way ANOVA. p<0.05 was considered significant. Results In the frontal cortex, only Fas-L expression was significantly lower in G-CSF group when compared with DHCA group. In the hippocampus, G-CSF increased Bcl-2 (54.3±6.4 vs. 32.3±2.2, p=0.001) and pAkt (141.4±19 vs. 95.9±21.1, p=0.047) when compared to DHCA group. Caspase-3, Bax, Fas, Fas-L, DR6 and pJAK2 levels were unchanged. The Bcl-2/Bax ratio was 0.33 for DHCA and 0.93 for G-CSF groups (p=0.02). In the striatum, when compared to DHCA group, G-CSF group had higher levels of Bcl-2 (50.3±7.4 vs. 31.8±3.8, p=0.01), pAkt (132.7±12.3 vs. 14±1.34, p=2.3×106), pJAK2 (126±17.4 vs.77.9±13.6, p=0.011), and lower Caspase-3 (12.8±5.0 vs. 32.2±11.5, p=0.033), Fas (390±31 vs. 581±74, p=0.038), Fas-L (20.5±11.5 vs. 57.8±15.6, p=0.04) and DR6 (57.4± 4.4 vs. 108.8±13.4, p=0.007). The Bcl-2/Bax ratio was 0.25 for DHCA and 0.44 for G-CSF groups (p=0.046). Conclusions In the piglet model of hypoxic brain injury, G-CSF decreases pro-apoptotic signaling, particularly in the striatum.

Objective: To determine whether the leaflets of bicuspid aortic valve (BAV) experience increased strain when compared to tricuspid aortic valve (TAV) leaflets. Background: The population at highest risk of aortic valve calcification (AVC) are individuals with BAVs. Currently, efforts to medically treat AVC are hampered by a limited understanding of the biomechanical forces involved in the molecular pathogenesis of AVC. Methods: Surgically created BAVs and control TAVs were placed into a left heart simulator. Strains were calculated by comparing the distances between points on the aortic valve (AoV) leaflet during various time points during a simulated cardiac cycle. Results: The fused leaflets of BAVs experience significantly more strain during systole when compared to TAVs. Specifically, BAVs experience 24% strain (P < .0001) in the radial direction, parallel to the direction of blood flow, as compared to TAVs. There was peak difference of 4% (P < .001) in the circumferential direction. Discussion: Based upon the data presented here, we are in the process of identifying how increased strain activates calcification-associated pathways in AoV cells. Future studies will examine whether these stretch responsive pathways can be blocked to inhibit calcification of BAVs.

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an “in vivo-like” environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.