Hirotsugu Kurobe

Coronary Atherosclerosis Is Associated With Macrophage Polarization in Epicardial Adipose Tissue

OBJECTIVES The purpose of this report was to assess the link between macrophage polarization in epicardial adipose tissue and atherosclerosis in patients with coronary artery disease (CAD). BACKGROUND Macrophage accumulation enhances chronic inflammation in adipose tissue, but macrophage phenotypic change in human epicardial adipose tissue and its role in atherogenesis are unknown. METHODS Samples were obtained from epicardial and subcutaneous adipose tissue during elective cardiac surgery (CAD, n = 38; non-CAD, n = 40). Infiltration of M1/M2 macrophages was investigated by immunohistochemical staining with antibodies against CD11c and CD206, respectively. Expression of pro- and anti-inflammatory adipocytokines in adipose tissue was evaluated by real-time quantitative polymerase chain reaction. RESULTS Infiltration of macrophages and expression of pro- and anti-inflammatory cytokines were enhanced in epicardial fat of patients with CAD compared with that in non-CAD patients (p < 0.05). The ratio of M1/M2 macrophages was positively correlated with the severity of CAD (r = 0.312, p = 0.039). Furthermore, the expression of pro-inflammatory cytokines was positively correlated, and the expression of anti-inflammatory cytokines was negatively correlated with the ratio of M1/M2 macrophages in epicardial adipose tissue of CAD patients. By contrast, there was no significant difference in macrophage infiltration and cytokine expression in subcutaneous adipose tissue between the CAD and non-CAD groups. CONCLUSIONS The ratio of M1/M2 macrophages in epicardial adipose tissue of CAD patients is changed compared with that in non-CAD patients. Human coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue.

Concise Review: Tissue-Engineered Vascular Grafts for Cardiac Surgery: Past, Present, and Future

In surgical repair for heart or vascular disease, it is often necessary to implant conduits or correct tissue defects. The most commonly used graft materials to date are (a) artificial grafts; (b) autologous tissues, such as pericardium and saphenous vein; (c) allografts; and (d) xenografts. However, none of these four options offer growth potential, and all are associated with varying levels of thrombogenicity and susceptibility to infection. The lack of growth potential of these four options is particularly important in pediatric cardiac surgery, where patients will often outgrow their vascular grafts and require additional operations. Thus, developing a material with sufficient durability and growth potential that will function as the child grows older will eliminate the need for reoperation and significantly reduce morbidity and mortality of some types of congenital heart defects. Vascular tissue engineering is a relatively new field that has undergone enormous growth over the last decade. The goal of vascular tissue engineering is to produce neovessels and neo‐organ tissue from autologous cells using a biodegradable polymer as a scaffold. The most important advantage of tissue‐engineered implants is that these tissues can grow, remodel, rebuild, and respond to injury. Once the seeded autologous cells have deposited an extracellular matrix and the original scaffold is biodegraded, the tissue resembles and behaves as native tissue. When tissue‐engineered vascular grafts are eventually put to use in the clinical arena, the quality of life in patients after surgery will be drastically improved.

Epicardial Adipose Tissue Volume and Adipocytokine Imbalance Are Strongly Linked to Human Coronary Atherosclerosis

Objective—The impact of epicardial adipose tissue (EAT) over abdominal or overall adiposity on coronary artery disease (CAD) is currently unknown. We compared the association among EAT volume (EATV), cytokine/adipocytokine profiles in EAT and subcutaneous fat, and atherogenic CAD. Approach and Results—Paired samples were obtained from EAT and subcutaneous adipose tissue during elective cardiac surgery for CAD (n=50) or non-CAD (n=50). EATV was the sum of cross-sectional EAT areas, and visceral and subcutaneous fat areas were determined at the umbilicus level on computed tomography scans. CD68+, CD11c+, and CD206+ cells were counted using immunohistochemical staining. Cytokine/adipocytokine expression was evaluated using quantitative real-time polymerase chain reaction. Multivariate analysis indicated that male sex, age, diabetes mellitus, high triglycerides, and low high-density lipoprotein cholesterol, and EATV index (EATV/body surface area, cm3/m2) were significant CAD predictors (corrected R2=0.401; P<0.001); visceral fat area, hypertension, smoking, low-density lipoprotein cholesterol (140 mg/dL [3.63 mmol/L]) or statin use were not predictors. The EATV index positively correlated with the CD68+ and CD11c+ cell numbers and nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3), interleukin-1&bgr;, and interleukin-1R expression; and negatively correlated with adiponectin expression in EAT. A multivariate analysis model, including CD68+ cells and interleukin-1&bgr;, and adiponectin expression in EAT strongly predicted CAD (corrected R2=0.756; P<0.001). Conclusions—EATV and macrophage and cytokine/adipocytokine signals in EAT strongly correlated with CAD. Our findings suggest that EATV and adipocytokine imbalance are strongly linked to human coronary atherosclerosis.

Telmisartan ameliorates insulin sensitivity by activating the AMPK/SIRT1 pathway in skeletal muscle of obese db/db mice

BackgroundTelmisartan is a well-established angiotensin II type 1 receptor blocker that improves insulin sensitivity in animal models of obesity and insulin resistance, as well as in humans. Telmisartan has been reported to function as a partial agonist of the peroxisome proliferator-activated receptor (PPAR) γ, which is also targeted by the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase (SIRT1). Here, we investigated the pathways through which telmisartan acts on skeletal muscle, in vitro as well as in vivo.MethodsNine-week-old male db/db mice were fed a 60% high-fat diet, with orally administrated either vehicle (carboxymethyl-cellulose, CMC), 5 mg/kg telmisartan, or 5 mg/kg telmisartan and 1 mg/kg GW9662, a selective irreversible antagonist of PPARγ, for 5 weeks. Effects of telmisartan on Sirt1 mRNA, AMPK phosphorylation, and NAD+/NADH ratio were determined in C2C12 cultured myocytes.Results and discussionTelmisartan treatment improved insulin sensitivity in obese db/db mice fed a high-fat diet and led to reduction in the size of hypertrophic pancreatic islets in these mice. Moreover, in vitro treatment with telmisartan led to increased expression of Sirt1 mRNA in C2C12 skeletal muscle cells; the increase in Sirt1 mRNA in telmisartan-treated C2C12 myoblasts occurred concomitantly with an increase in AMPK phosphorylation, an increase in NAD+/NADH ratio, and increases in the mRNA levels of PGC1α, FATP1, ACO, and GLUT4.ConclusionsOur results indicate that telmisartan acts through a PPARγ-independent pathway, but at least partially exerts its effects by acting directly on skeletal muscle AMPK/SIRT1 pathways.

UNDIMINISHED REGULATORY T CELLS IN THE THYMUS OF PATIENTS WITH MYASTHENIA GRAVIS

Objective: The thymus has been implicated as a possible site of origin that triggers autoimmunity in myasthenia gravis (MG). Although several groups have suggested that the decrease in the number of regulatory T (Treg) cells contributes to the onset of MG, the exact role of Treg cells in MG remains unclear. To address this point, we examined the number and distribution of Treg cells in a large number of patients with MG. Methods: Immunohistofluorescence analysis of Foxp3 along with CD4 and CD8 was performed in thymic sections of MG (+) (n = 24) and MG (−) patients (n = 27). Circulating CD4+CD25+ cells in the peripheral blood of patients with MG (n = 15) and age-matched healthy subjects (n = 15) were also analyzed. Results: Foxp3+CD4+CD8− cells were predominantly found in the thymic medulla and their number declined with age. There was no significant difference in the number or the distribution of Foxp3+CD4+CD8− cells in the thymus between MG (+) and MG (−) patients. The number of circulating CD4+CD25+ cells in the peripheral blood of patients with MG was not significantly altered compared to that in healthy subjects. Conclusion: The cellularity of Treg cells in the thymus and circulation is not diminished in patients with myasthenia gravis.

Exendin-4, a glucagon-like peptide-1 receptor agonist, attenuates neointimal hyperplasia after vascular injury

Exendin-4 is a glucagon-like peptide-1 receptor agonist that has been used as a drug for treatment of type 2 diabetes. To investigate the effect of exendin-4 on the cardiovascular system, we investigated the impact of exendin-4 on neointimal hyperplasia of the femoral artery after vascular injury. We performed wire-mediated endovascular injury in C57BL/6 mice, followed by administration of exendin-4 24 nmol/kg/day via infusion pump. Four weeks after the injury, exendin-4 treatment significantly attenuated neointimal hyperplasia of the injured artery, although it did not affect glucose metabolism and lipid profile in wild-type mice. Immunofluorescence study revealed abundant expression of GLP-1 receptor on α-smooth muscle actin-positive cells in the injured vessel. Cell proliferation assay using rat aortic smooth muscle cells showed that exendin-4 reduced PDGF-BB induced smooth muscle cell proliferation through the cAMP/PKA pathway. Exendin-4 also inhibited TNFα production by peritoneal macrophages in response to inflammatory stimulus. Our findings indicate that a GLP-1 receptor agonist attenuated neointimal formation after vascular injury. GLP-1 receptor agonists or drugs that raise endogenous GLP-1 level might be effective in the treatment of vascular diseases.

Gender disparities in the association between epicardial adipose tissue volume and coronary atherosclerosis: A 3-dimensional cardiac computed tomography imaging study in Japanese subjects

BackgroundGrowing evidence suggests that epicardial adipose tissue (EAT) may contribute to the development of coronary artery disease (CAD). In this study, we explored gender disparities in EAT volume (EATV) and its impact on coronary atherosclerosis.MethodsThe study population consisted of 90 consecutive subjects (age: 63 ± 12 years; men: 47, women: 43) who underwent 256-slice multi-detector computed tomography (MDCT) coronary angiography. EATV was measured as the sum of cross-sectional epicardial fat area on CT images, from the lower surface of the left pulmonary artery origin to the apex. Subjects were segregated into the CAD group (coronary luminal narrowing > 50%) and non-CAD group.ResultsEATV/body surface area (BSA) was higher among men in the CAD group than in the non-CAD group (62 ± 13 vs. 33 ± 10 cm3/m2, p < 0.0001), but did not differ significantly among women in the 2 groups (49 ± 18 vs. 42 ± 9 cm3/m2, not significant). Multivariate logistic analysis showed that EATV/BSA was the single predictor for >50% coronary luminal narrowing in men (p < 0.0001). Predictors excluded were age, body mass index, hypertension, diabetes mellitus, and hyperlipidemia.ConclusionsIncreased EATV is strongly associated with coronary atherosclerosis in men.

Evaluation of remodeling process in small-diameter cell-free tissue-engineered arterial graft

OBJECTIVE Autologous grafts are used to repair atherosclerotic cardiovascular diseases; however, many patients lack suitable donor graft tissue. Recently, tissue engineering techniques have emerged to make biologically active blood vessels. We applied this technique to produce arterial grafts using established biodegradable materials without cell seeding. The grafts were evaluated in vivo for vessel remodeling during 12 months. METHODS Poly(L-lactide-co-ε-caprolactone) scaffolds reinforced by poly(lactic acid) (PLA) fiber were prepared as arterial grafts. Twenty-eight cell-free grafts were implanted as infrarenal aortic interposition grafts in 8-week-old female SCID/Bg mice. Serial ultrasound and micro computed tomography angiography were used to monitor grafts after implantation. Five grafts were harvested for histologic assessments and reverse transcription-quantitative polymerase chain reaction analysis at time points ranging from 4 months to 1 year after implantation. RESULTS Micro computed tomography indicated that most implanted mice displayed aneurysmal changes (three of five mice at 4 months, four of five mice at 8 months, and two of five mice at 12 months). Histologic assessments demonstrated extensive tissue remodeling leading to the development of well-circumscribed neovessels with an endothelial inner lining, a neointima containing smooth muscle cells and elastin, and a collagen-rich extracellular matrix. There were a few observed calcified deposits, located around residual PLA fibers at 12 months after implantation. Macrophage infiltration into the scaffold, as evaluated by F4/80 immunohistochemical staining, remained after 12 months and was focused mostly around residual PLA fibers. Reverse transcription-quantitative polymerase chain reaction analysis revealed that gene expression of Itgam, a marker for macrophages, and of matrix metalloproteinase 9 was higher than in native aorta during the course of 12 months, indicating prolonged inflammation (Itgam at 8 months: 11.75 ± 0.99 vs native aorta, P < .01; matrix metalloproteinase 9 at 4 months: 4.35 ± 3.05 vs native aorta, P < .05). CONCLUSIONS In this study, we demonstrated well-organized neotissue of cell-free biodegradable arterial grafts. Although most grafts experienced aneurysmal change, such findings provide insight into the process of tissue-engineered vascular graft remodeling and should allow informed rational design of the next generation of arterial grafts.

Well-organized neointima of large-pore poly(l-lactic acid) vascular graft coated with poly(l-lactic-co-ε-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(l-lactic acid) graft in a mouse aortic implantation model

OBJECTIVE Tissue engineering techniques have emerged that allow bioresorbable grafts to be implanted that restore function and transform into biologically active arteries. However, these implants are susceptible to calcification during the remodeling process. The objective of this study was to evaluate the role of pore size of bioabsorbable grafts in the development of calcification. METHODS Two types of grafts were prepared: a large-pore graft constructed of poly(L-lactic acid) (PLA) fibers coated with poly(L-lactide-co-ε-caprolactone) (PLCL) (PLA-PLCL), and a small-pore graft made of electrospun PLA nanofibers (PLA-nano). Twenty-eight PLA-PLCL grafts and twenty-five PLA-nano grafts were implanted as infra-renal aortic interposition conduits in 8-week-old female SCID/Bg mice, and followed for 12 months after implantation. RESULTS Large-pore PLA-PLCL grafts induced a well-organized neointima after 12 months, and Alizarin Red S staining showed neointimal calcification only in the thin neointima of small-pore PLA-nano grafts. At 12 months, macrophage infiltration, evaluated by F4/80 staining, was observed in the thin neointima of the PLA-nano graft, and there were few vascular smooth muscle cells (VSMCs) in this layer. On the other hand, the neointima of the PLA-PLCL graft was composed of abundant VSMCs, and a lower density of macrophages (F4/80 positive cells, PLA-PLCL; 68.1 ± 41.4/mm(2) vs PLA-nano; 188.3 ± 41.9/mm(2), p = 0.007). The VSMCs of PLA-PLCL graft expressed transcription factors of both osteoblasts and osteoclasts. CONCLUSION These findings demonstrate that in mouse arterial circulation, large-pore PLA-PLCL grafts created a well-organized neointima and prevented calcific deposition compared to small-pore, electrospun PLA-nano grafts.

Development of small diameter nanofiber tissue engineered arterial grafts.

The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.