P. S. Tsao

Dimethylarginine Dimethylaminohydrolase Overexpression Suppresses Graft Coronary Artery Disease

Background—Graft coronary artery disease (GCAD) is the leading cause of death after the first year of heart transplantation. The reduced bioavailability of endothelium-derived nitric oxide (NO) may play a role in endothelial vasodilator dysfunction and the structural changes that are characteristic of GCAD. A potential contributor to endothelial pathobiology is asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor. We hypothesized that lowering ADMA concentrations by dimethylarginine dimethylaminohydrolase (DDAH) overexpression in the recipient might suppress GCAD and long-term immune responses in murine cardiac allografts. Methods and Results—In one series, donor hearts of C-H-2bm12KhEg (H-2bm12) wild-type (WT) mice were heterotopically transplanted into C57BL/6 (H-2b) transgenic mice overexpressing human DDAH-I or WT littermates and procured after 4 hours of reperfusion (WT and DDAH-I recipients, n=6 each). In a second series, donor hearts were transplanted into DDAH-I–transgenic or WT mice and procured 30 days after transplantation (n=7 each). In DDAH-I recipients, plasma ADMA concentrations were lower, in association with reduced myocardial generation of superoxide anion (WT versus DDAH-I, 465.7±79.8 versus 173.4±32.3 &mgr;mol · L−1 · mg−1 · h−1; P=0.02), inflammatory cytokines, adhesion molecules, and chemokines. GCAD was markedly reduced in cardiac allografts of DDAH-I–transgenic recipients as assessed by luminal narrowing (WT versus DDAH, 79±2% versus 33±7%; P<0.01), intima-media ratio (WT versus DDAH, 1.1±0.1 versus 0.5±0.1; P<0.01), and the percentage of diseased vessels (WT versus DDAH, 100±0% versus 62±10%; P<0.01). Conclusions—Overexpression of DDAH-I attenuated oxidative stress, inflammatory cytokines, and GCAD in murine cardiac allografts. The effect of DDAH overexpression may be mediated by its reduction of plasma and tissue ADMA concentrations.

DNA methylation signatures of chronic low-grade inflammation are associated with complex diseases

BackgroundChronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation.ResultsWe performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P < 1.15 × 10–7) in the discovery panel of European ancestry and replicated (P < 2.29 × 10–4) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P < 8.47 × 10–5), ten (17%) CpG sites were associated with a nearby genetic variant (P < 2.50 × 10–3), and 51 (88%) were also associated with at least one related cardiometabolic entity (P < 9.58 × 10–5). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants.ConclusionWe have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation.

Human internal mammary artery (IMA) transplantation and stenting: a human model to study the development of in-stent restenosis.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches (1,2). The commonly used animal models include mice, rats, rabbits, and pigs (3-5). However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models (6,7). In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents. This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

220 INSULIN RESISTANT INDIVIDUALS MAY BE CHARACTERIZED BY A PREPONDERANCE OF SMALLER ADIPOCYTES

Purpose There is an increasing understanding that adipose tissue not only serves as an energy storage depot but also acts as a powerful endocrine organ. Various studies have shown that important factors such as leptin, adiponectin, and resistin are not only produced in adipose tissue, but may have significant systemic effects. Such factors have been shown to be differentially expressed in insulin resistant (IR) and insulin sensitive (IS) individuals. Prior studies have suggested that adipocytes are larger in IR individuals while other studies have suggested impaired adipogenesis in IR individuals compared to IS ones. Our study aimed to test the hypothesis that there are differences in adipocyte size between IR and IS individuals and that these differences are independent of body mass index (BMI) and may be related to varying levels of differentiation. Methods Human biopsies were obtained from subcutaneous adipose tissue depots of relatively healthy overweight or obese individuals (n=10) with similar body mass index (BMI) values (IR 32.3 ± 3.8 vs. IS 29.4 ± 2.9). Insulin sensitivity was determined using steady state plasma glucose (SSPG) measurements to establish whether individuals were placed in the IR or IS group (IR 237 ± 42 vs. IS 77 ± 16 mg/dL). Adipocyte size was determined using a Multisizer Coulter Counter and were confirmed to be adipocytes by light microscopy. Gene expression levels were determined by quantitative real-time PCR. Results Although adipocyte size measurements between IR and IS groups did not differ significantly, the IR group had a higher small to large cell ratio (1.66± 1.03 vs. 0.94 ± 0.50, P ≤0.05). Adiponectin, PPARγ1, PPARγ2, and GLUT4 all showed 2-3 fold lower levels of expression in the IR group (n=5) compared to the IS group (n=5). In contrast, there were no significant differences in Adipsin and SREBP-1c expression in both groups. Conclusions Our observation of a bimodal distribution of small and large cells in both IR and IS groups, and a larger ratio of small adipocytes in IR compared to IS individuals is a novel finding. Gene expression analysis suggests that there is probably a decrease in differentiation since GLUT4 and Adiponectin, known markers of terminal adipocyte differentiation, were significantly decreased in the IR group. However since SREBP-1c and Adipsin were not differentially expressed in both groups more work will need to be done to further characterize the adipocyte differences in the IR and IS groups and to determine the significance of adipocyte size and insulin resistance status.