Akira Marui

Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration

To realize cardiac regeneration using human induced pluripotent stem cells (hiPSCs), strategies for cell preparation, tissue engineering and transplantation must be explored. Here we report a new protocol for the simultaneous induction of cardiomyocytes (CMs) and vascular cells [endothelial cells (ECs)/vascular mural cells (MCs)], and generate entirely hiPSC-engineered cardiovascular cell sheets, which showed advantageous therapeutic effects in infarcted hearts. The protocol adds to a previous differentiation protocol of CMs by using stage-specific supplementation of vascular endothelial cell growth factor for the additional induction of vascular cells. Using this cell sheet technology, we successfully generated physically integrated cardiac tissue sheets (hiPSC-CTSs). HiPSC-CTS transplantation to rat infarcted hearts significantly improved cardiac function. In addition to neovascularization, we confirmed that engrafted human cells mainly consisted of CMs in >40% of transplanted rats four weeks after transplantation. Thus, our HiPSC-CTSs show promise for cardiac regenerative therapy.

Pluripotent Stem Cell‐Engineered Cell Sheets Reassembled with Defined Cardiovascular Populations Ameliorate Reduction in Infarct Heart Function Through Cardiomyocyte‐Mediated Neovascularization

Although stem cell therapy is a promising strategy for cardiac restoration, the heterogeneity of transplanted cells has been hampering the precise understanding of the cellular and molecular mechanisms. Previously, we established a cardiovascular cell differentiation system from mouse pluripotent stem cells, in which cardiomyocytes (CMs), endothelial cells (ECs), and mural cells (MCs) can be systematically induced and purified. Combining this with cell sheet technology, we generated cardiac tissue sheets reassembled with defined cardiovascular populations. Here, we show the potentials and mechanisms of cardiac tissue sheet transplantation in cardiac function after myocardial infarction (MI). Transplantation of the cardiac tissue sheet to a rat MI model showed significant and sustained improvement of systolic function accompanied by neovascularization. Reduction of the infarct wall thinning and fibrotic length indicated the attenuation of left ventricular remodeling. Cell tracing with species‐specific fluorescent in situ hybridization after transplantation revealed a relatively early loss of transplanted cells and an increase in endogenous neovascularization in the proximity of the graft, suggesting an indirect angiogenic effect of cardiac tissue sheets rather than direct CM contributions. We prospectively dissected the functional mechanisms with cell type‐controlled sheet analyses. Sheet CMs were the main source of vascular endothelial growth factor. Transplantation of sheets lacking CMs resulted in the disappearance of neovascularization and subsequent functional improvement, indicating that the beneficial effects of the sheet were achieved by sheet CMs. ECs and MCs enhanced the sheet functions and structural integration. Supplying CMs to ischemic regions with cellular interaction could be a strategic key in future cardiac cell therapy. STEM CELLS2012;30:1196–1205

Angiogenic properties of sustained release platelet-rich plasma: Characterization in-vitro and in the ischemic hind limb of the mouse

BACKGROUND While single growth factor has limitation to induce optimal neovascularization, platelet-rich plasma (PRP) is an autologous reserver of various growth factors. However, little is known about the mechanism of PRP-related neovascularization.The objective of this investigation was to characterize the angiogenic and growth factor content of PRP and to determine, in vitro, its effect on endothelial cell proliferation. Additionally, this experiment sought to determine the effectiveness of different compositions of PRP (solution versus sustained release) on perfusion and neovascularization in a murine model of hind limb ischemia. METHODS Different growth factors were measured by enzyme-linked immunosorbent assay (ELISA). In vivo study, we used gelatin hydrogel as a sustained release carrier for growth factors in PRP. We induced hind limb ischemia by excising right femoral artery in wild type C57BL6 mice. After surgery, mice were randomly assigned to four experimental groups; control (C), 100 muL of sustained release form of platelet-poor plasma (PPP), 100 muL of solution form of PRP (PRP-sol), 100 muL of sustained release form of PRP (PRP-sr); each formulation was administered via an intramuscular injection to the ischemic hind limb. Endpoint evaluations were blood perfusion by laser Doppler perfusion image, vascular density by anti Von Willebrand factor (vWF), and mature vessel density by anti smooth muscle actin (SMA) antibody. Green fluorescent protein (GFP+) transgenic mice were generated by transplantation of bone marrow derived mononuclear cells to wild type C57BL6 mice, and finally CD34+ cell in the ischemic site of transgenic mice was detected by staining with anti-CD34 antibody. RESULTS In vitro study showed that PRP containing different growth factors induces endothelial cell proliferation and capillary tube formation. In vivo study demonstrated that sustained release of PRP increased perfusion of ischemic tissue as measured by laser Doppler perfusion imaging (LDPI) (57 +/- 12, 56 +/- 9, 72 +/- 7, 98 +/- 4 for groups C, PPP, PRP-sol, and PRP-sr, respectively; P < .05); capillary density (151 +/- 16, 158 +/- 12, 189 +/- 39, 276 +/- 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) and mature vessel density (28 +/- 2, 31 +/- 3, 52 +/- 10, 85 +/- 13 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) . Sustained release PRP also increases CD34+ cells in the ischemic site of transgenic mice (6 +/- 3 vs 18 +/- 5/mm(2) for groups control and PRP-sr respectively, P < .05). CONCLUSION Sustained release of PRP containing potent angiogenic growth factors restores blood perfusion presumably by stimulating angiogenesis, arteriogenesis, as well as vasculogenesis in the mouse hind limb ischemia. CLINICAL RELEVANCE PRP is a natural reserver of various growth factors that can be collected autologously and is costeffective. Thus for clinical use, no special considerations concerning antibody formation and infection risk are needed.Some clinical devices to automatically prepare PRP are available at present. PRP are consistently being used clinically inthe department of orthopedics and plastic surgery (oral, maxillary facial) for a long time. On the basis of researchevidence, some publications have reported positive results in either bone or soft tissue healing. However, some researchconcludes that there is no or little benefit from PRP. This is likely due to faster degradation of growth factors in PRP sincesome authors suggest using sustained release form of PRP to deliver optimal effect of PRP. Gelatin hydrogel is also beingused clinically as a slow, sustained release of carrier for growth factors in our center recently.

Therapeutic angiogenesis by the controlled release of basic fibroblast growth factor for ischemic limb and heart injury: toward safety and minimal invasiveness.

We review our studies on therapeutic angiogenesis using basic fibroblast growth factor (bFGF) released in a controlled manner from biodegradable gelatin hydrogel (GH). The bFGF-GH was intramuscularly injected in rabbits with limb ischemia. The group treated with bFGF showed an increase in tissue blood flow under laser Doppler imaging and histology showed a greater vascular density compared with controls. Also, bFGF-GH was subepicardially injected into old heart infarcts in rats. In the group treated with bFGF, improved left ventricular function was shown by echocardiography and cardiac catheterization, increased regional blood flow in the peri-infarct area was detected by pinhole single-photon emission computed tomography using 201Tl, and increased vascular density was demonstrated by histology. In rabbits with acute myocardial infarction, the heart was wrapped with the omentum (including the gastroepiploic artery) and a bFGF-GH sheet was applied. Postoperative assessment revealed rich communication from the gastroepiploic artery to the coronary artery and improved cardiac function. The controlled release of bFGF was effective for both limb and heart ischemia and is considered to be suitable for clinical use because its application in animals was feasible and safe with minimal invasiveness.

Comparison of Contrast-Enhanced MRI with 18F-FDG PET/201Tl SPECT in Dysfunctional Myocardium: Relation to Early Functional Outcome After Surgical Revascularization in Chronic Ischemic Heart Disease

Revascularization of viable myocardial segments has been shown to improve left ventricular (LV) function and long-term prognosis; however, the surgical risk is comparatively higher in patients with a low ejection fraction (EF). We compared contrast-enhanced MRI with 18F-FDG PET/201Tl SPECT for myocardial viability and prediction of early functional outcome in patients with chronic coronary artery disease (CAD). Methods: Forty-one patients with chronic CAD and LV dysfunction (mean age ± SD, 66 ± 10 y; 32 men; mean EF ± SD, 38% ± 13%) referred for 18F-FDG PET, 201Tl-SPECT and MRI within 2 wk were included. Twenty-nine subjects underwent coronary artery bypass grafting (CABG), and LV function was reassessed by MRI before discharge (17 ± 7 d after surgery). Two were excluded from outcome analysis (1 death due to sepsis; 1 perioperative myocardial infarction). The extent of viable myocardium by 18F-FDG PET/201Tl SPECT was defined by the metabolism–perfusion mismatch or ischemia, in comparison with the extent of delayed enhancement (DE) on MRI in a 17-segment model. Segmental functional recovery was defined as improvement in the wall motion score of ≥1 on a 4-point scale. EF and LV volume change were used as global functional outcome. Results: Three hundred ninety-four dysfunctional segments were compared, and the extent of DE on MRI correlated negatively with the viability on 18F-FDG PET. Of 252 dysfunctional segments that were successfully revascularized, the sensitivity, specificity, positive predictive value, and negative predictive value of PET/SPECT were 60.2%, 98.7%, 76.6%, and 96.7% and of MRI were 92.2%, 44.9%, 72.4%, and 78.6% using the cutoff value of 50% DE on MRI, without significant differences in overall accuracies. In 18 subjects who underwent isolated CABG, improvement of EF (≥5%) and reverse LV remodeling (≥10% LV size reduction) was best predicted by the no DE on MRI, and patients with substantial nonviable myocardium on 18F-FDG/SPECT predicted a poor early functional outcome (all P < 0.001). Conclusion: Accurate prediction of early functional outcome by PET/SPECT and contrast-enhanced MRI is possible.

Enhanced angiogenesis by gelatin hydrogels incorporating basic fibroblast growth factor in rabbit model of hind limb ischemia

Recently we have developed new sustained release system of basic fibroblast growth factor (bFGF) using gelatin hydrogel as a carrier. Using this system, we examined the effect of topical sustained release of bFGF on angiogenesis and tissue blood perfusion in a rabbit model of hind limb ischemia. Thirty-two rabbits underwent excision of right femoral artery under general anesthesia. Two weeks later the rabbits were randomized into four groups (n = 8 each): no treatment, intramuscular injection of gelatin hydrogel alone, and intramuscular injection of gelatin hydrogel incorporating 30 µg and 100 µg of bFGF. Four weeks after each treatment, selective angiography, tissue blood flowmetry using laser Doppler perfusion imaging, and histological examination of thigh muscle were performed. In groups treated with bFGF incorporating gelatin hydrogel, tissue blood flow, number of arterioles, and vascular density were significantly increased in a dose-dependent manner 4 weeks after the treatment. Serum concentrations of bFGF and vascular endothelial growth factor were not elevated 4 weeks after the treatment. In conclusion, sustained release of bFGF using gelatin hydrogel augmented angiogenesis and improved tissue blood flow after excision of the femoral artery.

Benefits of Off-Pump Coronary Artery Bypass Grafting in High-Risk Patients

Background— The benefits of off-pump coronary artery bypass graft (OPCAB) compared with conventional on-pump coronary artery bypass graft (CCAB) remain controversial. Thus, it is important to investigate which patient subgroups may benefit the most from OPCAB rather than CCAB. Methods and Results— Among the patients undergoing first coronary revascularization enrolled in the CREDO-Kyoto Registry (a registry of first-time percutaneous coronary intervention and coronary artery bypass graft patients in Japan), 2468 patients undergoing coronary artery bypass graft were entered into the study (mean age, 67±9 years). Predicted risk of operative mortality (PROM) of each patient was calculated by logistic EuroSCORE. Patients were divided into tertile based on their PROM. Mortality rates and the incidences of cardiovascular events were compared between CCAB and OPCAB within each PROM tertile using propensity score analysis. A total of 1377 patients received CCAB whereas 1091 received OPCAB. Adjusted 30-day mortality was not significantly different between CCAB and OPCAB patients regardless of their PROM range. However, the odds ratio of 30-day stroke in CCAB compared with OPCAB in the high-risk tertile was 8.30 (95% confidence interval, 2.25–30.7; P<0.01). Regarding long-term outcomes, hazard ratio of stroke in CCAB compared with OPCAB in the high-risk tertile was 1.80 (95% confidence interval, 1.07–3.02; P=0.03). Nevertheless, hazard ratio of overall mortality in the high-risk tertile was 1.44 (95% confidence interval, 0.98–2.11; P=0.06), indicating no statistically significant difference between the 2 procedures. Conclusions— OPCAB as opposed to CCAB is associated with short-term and long-term benefits in stroke prevention in patients at higher risk as estimated by EuroSCORE. No survival benefit of OPCAB was shown regardless of preoperative risk level.

Left Ventricular Functional Analysis Using 64-Slice Multidetector Row Computed Tomography: Comparison with Left Ventriculography and Cardiovascular Magnetic Resonance

Objective: The progress in computed tomography (CT) has improved temporal resolution and shortened the acquisition time. We compared cardiac function using 64-slice CT with left ventriculography (LVG) and cardiovascular magnetic resonance (CMR). Methods: A head-to-head comparison between CT, LVG and CMR was performed in 41 patients. In global LV function, CMR served as the reference. Regional wall motion was compared in a 5-point scoring system. Results: CT had excellent intra- and interobserver reproducibility. Ejection fraction, end-diastolic and end-systolic volumes by CT were closely correlated with CMR (r = 0.95, 0.96 and 0.98, respectively), while LVG underestimated LV volumes (p < 0.01). The standard deviation of ejection fraction difference between CT and CMR was significantly lower than that between LVG and CMR (p = 0.0015). In regional function, there were good agreements of 94.8% (ĸ = 0.82) between CT and LVG and 94.5% (ĸ = 0.84) between CT and CMR. The intermethod agreements in mild hypokinesis using CT tended to be lower. Conclusion: An excellent correlation was observed between CT and CMR in the LV function over a wide range of heart rates. However, even though 64-slice CT tended to be less sensitive in detecting mild hypokinesis, it still showed excellent concordance in advanced regional abnormalities.

Impact of left atrial volume reduction concomitant with atrial fibrillation surgery on left atrial geometry and mechanical function

OBJECTIVE Left atrial geometry and mechanical functions exert a profound effect on left ventricular filling and overall cardiovascular performance. We sought to investigate the perioperative factors that influence left atrial geometry and mechanical functions after the Maze procedure in patients with refractory atrial fibrillation and left atrial enlargement. METHODS Seventy-four patients with atrial fibrillation and left atrial enlargement (diameter > or = 60 mm) underwent the Maze procedure in association with mitral valve surgery. The maximum left atrial volume and left atrial mechanical functions (booster pump, reservoir, and conduit function [%]) were calculated from the left atrial volume-cardiac cycle curves obtained by magnetic resonance imaging. A stepwise multiple regression analysis was performed to determine the independent variables that influenced the postoperative left atrial geometry and function. RESULTS The multivariate analysis showed that left atrial reduction surgery concomitant with the Maze procedure and the postoperative maintenance of sinus rhythm were predominant independent variables for postoperative left atrial geometry and mechanical functions. Among the 58 patients who recovered sinus rhythm, the postoperative left atrial geometry and function were compared between patients with (VR group) and without (control group) left atrial volume reduction. At a mean follow-up period of 13.8 months, sinus rhythm recovery rate was better (85% vs 68%, P < .05) in the VR group and maximum left atrial volume was less (116 +/- 25 mL vs 287 +/- 73 mL, P < .001) than in the control group. The maximum left atrial volume reduced with time only in the VR group (reverse remodeling). Postoperative booster pump and reservoir function in the VR group were better than in the control group (25% +/- 6% vs 11% +/- 4% and 34% +/- 7% vs 16% +/- 4%, respectively, P < .001), whereas the conduit function in the VR group was lower than in the control group, indicating that the improvement of the booster pump and reservoir function compensated for the conduit function to left ventricular filling. CONCLUSION Left atrial reduction concomitant with the Maze procedure helped restore both contraction (booster pump) and compliance (reservoir) of the left atrium and facilitated left atrial reverse remolding. Left atrial volume reduction and postoperative maintenance of sinus rhythm may be desirable in patients with refractory AF and left atrial enlargement.

Therapeutic Treatment with Sustained-Release Platelet-Rich Plasma Restores Blood Perfusion by Augmenting Ischemia-Induced Angiogenesis and Arteriogenesis in Diabetic Mice

Objective: The objective of this investigation was to establish the effectiveness of sustained-release platelet-rich plasma (PRP) on perfusion and neovascularization in diabetic murine hind limb ischemia. Methods: After surgery in streptozotocin-induced diabetic mice, the mice were randomly assigned to the following 4 experimental groups: control (C), 100 µl of the sustained-release form of platelet-poor plasma (PPP), 100 µl of the solution form of PRP (PRP-sol), and 100 µl of the sustained-release form of PRP (PRP-sr). Endpoint evaluations were: blood perfusion by laser Doppler perfusion imaging (LDPI), vascular density by anti-vWF, and mature vessel density by anti-smooth muscle actin antibody. Results: This study demonstrated that a sustained release of PRP increases the perfusion of ischemic tissue as measured by LDPI (57 ± 12; 56 ± 9; 72 ± 7, and 98 ± 4 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05), capillary density (151 ± 16; 158 ± 12; 189 ± 39, and 276 ± 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively; p < 0.05), and mature vessel density (28 ± 2; 31 ± 3; 52 ± 10, and 85 ± 13 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05). Conclusion: A sustained release of PRP containing potent angiogenic growth factors restores blood perfusion by stimulating angiogenesis and arteriogenesis.