Debashish Das

Genome-wide association study in individuals of South Asian ancestry identifies six new type 2 diabetes susceptibility loci

We carried out a genome-wide association study of type-2 diabetes (T2D) in individuals of South Asian ancestry. Our discovery set included 5,561 individuals with T2D (cases) and 14,458 controls drawn from studies in London, Pakistan and Singapore. We identified 20 independent SNPs associated with T2D at P < 10−4 for testing in a replication sample of 13,170 cases and 25,398 controls, also all of South Asian ancestry. In the combined analysis, we identified common genetic variants at six loci (GRB14, ST6GAL1, VPS26A, HMG20A, AP3S2 and HNF4A) newly associated with T2D (P = 4.1 × 10−8 to P = 1.9 × 10−11). SNPs at GRB14 were also associated with insulin sensitivity (P = 5.0 × 10−4), and SNPs at ST6GAL1 and HNF4A were also associated with pancreatic beta-cell function (P = 0.02 and P = 0.001, respectively). Our findings provide additional insight into mechanisms underlying T2D and show the potential for new discovery from genetic association studies in South Asians, a population with increased susceptibility to T2D.

24 Protecting the Heart at a Distance: Exosomes for nano-sized Cardioprotection

Rationale Preconditioning is widely known to protect cardiomyocytes from reperfusion-induced cell death by activation of several pro-survival transductional pathways. The fact that preconditioning can be achieved remotely (Remote Ischaemic Preconditioning, RIPC) means that humoral factors are released from ischaemic limbs into the circulation carrying a pro-survival message. Exosomes are circulating nano-sized vesicles that mediate inter-cellular communication by carrying diverse proteins and RNA molecules. Here we studied the role of exosomes in mediating RIPC. Methods and Results We isolated exosomes from plasma of rats or humans subjected to RIPC. We characterised control or RIPC exosomes by electron microscopy, flow cytometry, western blot and nano-particle tracking analysis. Exosome concentration increased dramatically after RIPC in humans (from 3.5 ± 0.3x108 to 1.1 ± 0.3x109 exosomes/ml plasma; p < 0.01, n = 6), and administration of purified exosomes protected the heart from infarct in different settings including an in vivo rat model (vehicle: 47.4 ± 4.7; RIPC-Exosomes: 20.5 ± 3.9%Infarct/AAR; p < 0.01), ex vivo Langendorff (vehicle: 35.2 ± 3.3; RIPC-Exosomes: 21.2 ± 2.5% Infarct/AAR; p < 0.01), and in vitro hypoxia-reoxygenation of cardiomyocytes (43 ± 7% protection from death, p < 0.01). RIPC-Exosomes triggered rapid ERK phosphorylation (3.9 ± 0.1 fold over vehicle), and inhibition of upstream PI3K or MEK abolished ERK activation and inhibited cardioprotection. Conclusions We demonstrate that RIPC dramatically increases the concentration of exosomes in the circulation. Exosomes acutely activate pro-survival kinases that rapidly prepare the heart against ischemia-reperfusion injury. Exosomes represent a novel agent with the potential to be an endogenous, non-immunogenic and multi-signalling tool for cardioprotection.

238 RAT PLASMA EXOSOMES ARE CARDIOPROTECTIVE

Background The restoration of blood and oxygen to ischaemic myocardium under threat of infarction is of paramount importance, but reperfusion paradoxically exacerbates injury (IR injury). Exosomes are extracellular, lipid bilayer vesicles that range from 30 to 100nm in diameter. Exosomes released from in vitro cultured stem cells have been shown to be cardioprotective. Exosomes are also present in the blood of healthy individuals, but their properties are unknown. We hypothesized that plasma exosomes have cardioprotective properties. Aim To characterize the circulating exosomes of healthy rats, and to determine whether they protect against IR injury in HL-1 cardiac cells, in a Langendorff isolated, perfused rat heart model, and in an in vivo rat model of IR injury. Methods and Results Exosomes were isolated from rat plasma using the standard ultracentrifugation protocol. Their identity as exosomes was confirmed by their typical diameter of 87±2 nm measured by nanoparticle tracking analysis, their typical-shape morphology by electron microscopy, and by their expression of exosome marker proteins CD63 and HSP70. An average concentration of 2.5±1.1x1011 exosomes per ml of rat plasma was measured (N=5 rats). Similar results were obtained with human exosomes. After in vitro IR injury, 21±5% of HL-1 cells remained alive. Addition of exosomes increased survival to 49±6%. Control perfused rat hearts subject to IR had infarct sizes of 35±3 % (N=6). Perfusion with purified exosomes significantly reduced infarct size after IR to 23±2 % (N=10,P<0.01). In the in vivo model, i.v. injection of exosomes reduced infarct size from 47±4 % to 24±11 % (N=3-6, P<0.01). Conclusion This is the first data showing that circulating endogenous exosomes from healthy rats are capable of conferring cardioprotection. This contrasts with data suggesting larger microvesicles in the blood are detrimental to the cardiovascular system. Disease states which alter exosome number might potentially alter innate cardiac resistance to IR injury. Possible mechanisms will be discussed.