Selected abstracts:
Variation in COMT and UGT1A6 is associated with extreme morphine responses in infants and children
Erika N Scott, Jia He Zhang, Rena M Daniel, Graeme Ernest-Hoar, Julia K Charlton, Alice van Zanten, Jessica M Lovnicki, Colin JD Ross, S Rod Rassekh, Bruce C Carleton, Ruth E Grunau, and Catrina M Loucks
Introduction: Morphine/fentanyl are commonly prescribed for relief of major pain in children; however, responses range from inadequate pain relief (requiring higher doses/other analgesics) to adverse effects (e.g., respiratory depression, hypotension, allergic reaction), and the long-term consequences of opioid use on the developing brain remain unclear. Several genes are associated with opioid response; however, inconsistent findings have hindered development of pharmacogenetic tests to predict undesirable responses. Importantly, patients with extreme opioid responses (adverse effects at low doses or no pain relief despite high doses) are likely to harbour genetic variants with large effects, increasing the ability to detect associations. Objective: To investigate biologically relevant genetic variants in extreme responders to ultimately improve opioid safety and effectiveness in children. Methods: 161 patients were recruited from the NICU and pediatric oncology departments at BC Children’s and Women’s Hospitals as well as pediatric oncology departments across Canada, comprising 70 patients with inadequate pain relief or adverse effects to opioids and 91 patients with typical opioid responses. Genome-wide genotyping was conducted on DNA collected from all patients, and variants previously associated with opioid exposure and response were investigated. Results: COMT (mechanistically involved in pain perception and opioid-based pain relief) rs4680 was more frequent in controls with typical opioid responses (odds ratio [95% confidence interval] = 0.42 [0.22-0.79]). Additionally, rs17863783 in the drug metabolizing enzyme, UGT1A6, was more frequent in patients with inadequate pain relief/adverse effects (odds ratio [95% confidence interval] = 4.21 [0.85-20.95]). Conclusion/Next Steps: Patients carrying COMT rs4680 might require lower opioid doses for effective pain relief, while patients carrying UGT1A6 rs17863783 might experience inadequate pain relief/adverse effects due to increased metabolism of opioids. These associations are promising, and further analyses and functional studies will help to verify the roles that these genetic variants play in opioid-based pain relief. Identification of genetic variants that contribute to extreme opioid responses will help facilitate development of predictive pharmacogenetic tests so that the safest and most effective pain management strategies are used.
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Novel Roles of Matrix Metalloproteinase-2 in Myocardial Ischemia-Reperfusion Injury
Wesam Bassiouni, John M. Seubert, Richard Schulz
Background: Myocardial ischemia-reperfusion (IR) injury is accompanied by enhanced oxidative stress and impaired mitochondrial respiration. Tethering of mitochondria to the endoplasmic reticulum is essential for maintaining mitochondrial quality and function and is regulated by different proteins including the mitochondrial fusion protein, mitofusin-2 . Matrix metalloproteinase-2 (MMP-2) is a multifunctional protease that cleaves both extracellular and intracellular proteins. It is rapidly activated intracellularly in response to oxidative stress along with the de novo expression of its N-terminal truncated isoform (NTT-MMP-2) which is localized at or near mitochondria. Here we investigated whether MMP-2 proteolyzes mitofusin-2 during IR injury and impairs mitochondrial respiration. Methods: Hearts from 3 month old C57BL/6J mice were isolated and subjected to IR injury in vitro (30 min ischemia/40 min reperfusion) in absence or presence of MMP-2 inhibitors ARP-100 (10 µM), ONO-4817 (50 µM) or DMSO (vehicle) (n=5 hearts per group). Permeabilized muscle fibers were isolated from hearts at the end of reperfusion and used for measuring mitochondrial respiration. mRNA levels of full length and NTT-Mmp-2 were measured. The level of mitofusin-2 in mitochondria-enriched fractions and its proteolysis by MMP-2 were also evaluated. Results: Hearts subjected to IR showed a significant reduction in left ventricular developed pressure (LVDP), together with reduced mitochondrial oxygen consumption rate (OCR) and ATP production in fibers isolated from IR compared to aerobic hearts. ARP-100 or ONO-4817 improved post-ischemic recovery of LVDP and mitochondrial respiratory function compared to vehicle-perfused IR hearts. mRNA expression of NTT-Mmp-2 was significantly increased in IR hearts compared to aerobic controls. IR also significantly reduced mitofusin-2 protein level, while ARP-100 or ONO-4817 attenuated this reduction. Proteolysis of endogenous mitofusin- 2 in mitochondria-enriched fractions obtained from aerobic hearts was shown upon incubation with recombinant MMP-2, while ARP-100 prevented its cleavage. Conclusions: MMP-2 activated during IR injury contributes to the reduced cardiac contractile function and impaired mitochondrial respiration which could be attributed, at least in part, to the cleavage of mitofusin-2 by MMP-2. Inhibition of MMP-2 activity could protect against cardiac and mitochondrial dysfunction during IR injury.
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Therapeutic targeting of IL-10 signaling alleviates capillary stalling and cognitive impairment in Type 1 diabetes
Sorabh Sharma, Manjinder Cheema, Roobina Boghozian, Kelly A Tennant, Craig E Brown
Type 1 diabetes (T1D) has been associated with impaired cognitive function and vascular pathology. However, whether T1D perturbs blood flow in capillaries and what mechanisms mediate this phenomenon, remain elusive. Here, we longitudinally imaged the cerebral microcirculation in the streptozotocin model of T1D. Imaging revealed an increase in the number of stalled capillaries in the somatosensory cortex of T1D mice. Stalled capillaries were primarily associated with RBCs rather than leukocytes. These in vivo findings were validated by i.v injection of microspheres, which indicated significantly higher levels of capillary obstructions in diabetic mice. Behaviourally, diabetic mice were not different from controls in tests of ambulatory activity and visual function. However, diabetic mice were significantly impaired in learning/memory tests. In order to provide a mechanistic explanation, we probed for multiple cytokines and found unusually high levels of IL-10 in diabetic blood plasma. We then manipulated the IL10RA receptors broadly or in a cell specific manner. Interestingly, we found that only the endothelial specific knockdown of IL10RA receptors reduced the number of obstructed capillaries in diabetic mice but not the neutrophil specific IL10RA receptor knockdown. To test a clinically relevant approach, we injected IL-10RA neutralizing antibodies and found this was highly effective in preventing stalled capillaries in diabetic mice. Furthermore, chronic treatment of diabetic mice with IL10RA neutralizing antibodies led to significant improvements in CBF and cognitive function. In conclusion, our data indicates that diabetes is associated with greater risk for capillary obstructions as well as learning/memory deficits and blocking the IL10Ra receptors could be a therapeutic strategy to rescue such effects.
