Torsade de Pointes (TdP) is a lethal arrhythmia that is often drug-induced, thus there is an urgent need for development of models to test or predict the drug sensitivity of human cardiac tissue. ...Here, we present an in vitro TdP model using 3D cardiac tissue sheets (CTSs) that contain a mixture of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and non-myocytes. We simultaneously monitor the extracellular field potential (EFP) and the contractile movement of the CTSs. Upon treatment with IKr channel blockers, CTSs exhibit tachyarrhythmias with characteristics of TdP, including both a typical polymorphic EFP and meandering spiral wave re-entry. The TdP-like waveform is predominantly observed in CTSs with the cell mixture, indicating that cellular heterogeneity and the multi-layered 3D structure are both essential factors for reproducing TdP-like arrhythmias in vitro. This 3D model could provide the mechanistic detail underlying TdP generation and means for drug discovery and safety tests.
Abstract Background Current guidelines generally recommend watchful waiting until symptoms emerge for aortic valve replacement (AVR) in asymptomatic patients with severe aortic stenosis (AS). ...Objectives The study sought to compare the long-term outcomes of initial AVR versus conservative strategies following the diagnosis of asymptomatic severe AS. Methods We used data from a large multicenter registry enrolling 3,815 consecutive patients with severe AS (peak aortic jet velocity >4.0 m/s, or mean aortic pressure gradient >40 mm Hg, or aortic valve area <1.0 cm2 ) between January 2003 and December 2011. Among 1,808 asymptomatic patients, the initial AVR and conservative strategies were chosen in 291 patients, and 1,517 patients, respectively. Median follow-up was 1,361 days with 90% follow-up rate at 2 years. The propensity score–matched cohort of 582 patients (n = 291 in each group) was developed as the main analysis set for the current report. Results Baseline characteristics of the propensity score–matched cohort were largely comparable, except for the slightly younger age and the greater AS severity in the initial AVR group. In the conservative group, AVR was performed in 41% of patients during follow-up. The cumulative 5-year incidences of all-cause death and heart failure hospitalization were significantly lower in the initial AVR group than in the conservative group (15.4% vs. 26.4%, p = 0.009; 3.8% vs. 19.9%, p < 0.001, respectively). Conclusions The long-term outcome of asymptomatic patients with severe AS was dismal when managed conservatively in this real-world analysis and might be substantially improved by an initial AVR strategy. (Contemporary Outcomes After Surgery and Medical Treatment in Patients With Severe Aortic Stenosis Registry; UMIN000012140 )
Blood vessels are essential components for many tissues and organs. Thus, efficient induction of endothelial cells (ECs) from human pluripotent stem cells is a key method for generating higher tissue ...structures entirely from stem cells. We previously established an EC differentiation system with mouse pluripotent stem cells to show that vascular endothelial growth factor (VEGF) is essential to induce ECs and that cyclic adenosine monophosphate (cAMP) synergistically enhances VEGF effects. Here we report an efficient and robust EC differentiation method from human pluripotent stem cell lines based on a 2D monolayer, serum-free culture. We controlled the direction of differentiation from mesoderm to ECs using stage-specific stimulation with VEGF and cAMP combined with the elimination of non-responder cells at early EC stage. This "stimulation-elimination" method robustly achieved very high efficiency (>99%) and yield (>10 ECs from 1 hiPSC input) of EC differentiation, with no purification of ECs after differentiation. We believe this method will be a valuable technological basis broadly for regenerative medicine and 3D tissue engineering.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Objective Inflammation-mediated elastin destruction in the aortic medial layer is related to progression of abdominal aortic aneurysm (AAA). Epigallocatechin-3-gallate (EGCG), a major ...component of green tea polyphenols, reportedly increases elastin synthesis in vitro and may possess anti-inflammatory effects. We used a rat model to investigate whether EGCG could prevent AAA progression. Methods AAA was induced with administration of intraluminal elastase and extraluminal CaCl2 in male rats. Rats were randomly divided into a control group (n = 30) and an EGCG group (n = 30). In the EGCG group, an EGCG solution (20 mg/d) was administered orally to each rat from 2 weeks before AAA induction and continued 4 weeks beyond induction. Results The abdominal aortic diameter was significantly smaller in the EGCG group than in the control group on day 28 (2.9 ± 0.2 vs 2.3 ± 0.1 mm; P < .0001). The medial layer wall thickness and elastin content were significantly greater in the EGCG group than in the control group on day 28 (68.4 ± 13.6 vs 46.7 ± 13.4 μm P < .001 and 20.3 ± 4.6 vs 9.5 ± 3.6% P < .0001, respectively). Gene expression levels of tropoelastin and lysyl oxidase were significantly higher in the EGCG group immediately before AAA induction, indicating promoted elastoregeneration by EGCG administration (tropoelastin: 0.59 ± 0.36 control vs 1.24 ± 0.36 EGCG P < .05, lysyl oxidase: 0.77 ± 0.45 control vs 1.34 ± 0.4 EGCG P < .05) (fold increase). Gene expression levels of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, were significantly downregulated in the EGCG group (1.82 ± 0.71 vs 0.97 ± 0.59 P < .05 and 3.91 ± 3.24 vs 0.89 ± 0.59 P < .05, respectively). On day 7, gene expression levels and gelatinolytic activity of matrix metalloproteinase 9 were significantly lower in the EGCG group (1.41 ± 0.86 vs 0.51 ± 0.42 P < .05 and 1.00 ± 0.17 vs 0.29 ± 0.12 P < .0001, respectively), whereas gene expression levels of tissue inhibitors of metalloproteinase-1 were significantly higher in the EGCG group (0.96 ± 0.11 vs 1.14 ± 0.09; P < .05). Conclusions EGCG attenuated AAA progression in a rat model by preserving the aortic thickness and elastin content of the medial layer through regeneration of elastin, as mediated by anti-inflammatory effects, and subsequent reduction of matrix metalloproteinase activity.
To realize human induced pluripotent stem cell (hiPSC)-based cardiac regenerative therapy, evidence of therapeutic advantages in human-sized diseased hearts are indispensable. In combination with an ...efficient and simultaneous differentiation of various cardiac lineages from hiPSCs and cell sheet technology, we aimed to generate clinical-sized large cardiac tissue sheets (L-CTSs) and to evaluate the therapeutic potential in porcine infarct heart. We simultaneously induced cardiomyocytes (CMs) and vascular cells vascular endothelial cells (ECs) and vascular mural cells (MCs) from hiPSCs. We generated L-CTSs using 10cm-sized temperature-responsive culture dishes. We induced myocardial infarction (MI) in micromini-pigs (15-25 kg) and transplanted the L-CTSs (Tx) 2 weeks after MI induction (4 sheets/recipient) under immunosuppression (Tx: n = 5, Sham: n = 5). Self-pulsating L-CTSs were approximately 3.5cm in diameter with 6.8×106±0.8 of cells containing cTnT+-CMs (45.6±13.2%), VE-cadherin+-ECs (5.3±4.4%) and PDGFRβ+-MCs (14.4±20.7%), respectively (n = 5). In Tx group, echocardiogram indicated a significantly higher systolic function of the left ventricle (LV) compared to that in sham control (Sham vs Tx: fractional shortening: 24.2±8.6 vs 40.5±9.7%; p<0.05). Ejection fraction evaluated by left ventriculogram was significantly higher in Tx group (25.3±6.2% vs 39.8±4.2%; p<0.01). Speckle tracking echocardiogram showed a significant increase of circumference strain in infarct and border regions after transplantation. Fibrotic area was significantly lower in Tx group (23.8±4.5 vs 15.9±3.8%; P<0.001). Capillary density in the border region was significantly higher in Tx group (75.9±42.6/mm2 vs 137.4±44.8/mm2, p<0.001). These data indicate that the L-CTS transplantation attenuated LV remodeling. L-CTSs potentially restore cardiac dysfunction of human-sized infarct heart.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human induced pluripotent stem cells (hiPSCs) are a robust source for cardiac regenerative therapy due to their potential to support autologous and allogeneic transplant paradigms. The in vitro ...generation of three-dimensional myocardial tissue constructs using biomaterials as an implantable hiPSC-derived myocardium provides a path to realize sustainable myocardial regeneration. We generated engineered cardiac tissues (ECTs) from three cellular compositions of cardiomyocytes (CMs), endothelial cells (ECs), and vascular mural cells (MCs) differentiated from hiPSCs. We then determined the impact of cell composition on ECT structural and functional properties. In vitro force measurement showed that CM+EC+MC ECTs possessed preferential electromechanical properties versus ECTs without vascular cells indicating that incorporation of vascular cells augmented tissue maturation and function. The inclusion of MCs facilitated more mature CM sarcomeric structure, preferential alignment, and activated multiple tissue maturation pathways. The CM+EC+MC ECTs implanted onto infarcted, immune tolerant rat hearts engrafted, displayed both host and graft-derived vasculature, and ameliorated myocardial dysfunction. Thus, a composition of CMs and multiple vascular lineages derived from hiPSCs and incorporated into ECTs promotes functional maturation and demonstrates myocardial replacement and perfusion relevant for clinical translation.
Abstract Background Cardiopulmonary bypass (CPB) may induce systemic inflammatory responses causing acute lung injury (ALI). Recombinant human soluble thrombomodulin (rTM) is reported to attenuate ...the secretion of inflammatory cytokines and the high-mobility group box 1 (HMGB1) protein, which is critical in controlling systemic inflammation and apoptosis. We investigated the protective effects of rTM on CPB-induced lung injury in a rat model. Methods Eighteen male, Sprague–Dawley rats were divided into three groups: Sham, control (CPB alone), and rTM (CPB + rTM). CPB was conducted in the control group and the rTM group. A bolus of rTM (3 mg/kg) was administered to the rTM group rats before CPB establishment. Results The PaO2 /FiO2 ratio only dropped markedly from before CPB in the control group (p < 0.001). Serum TNF-α, IL-6, and HMGB1 levels were significantly higher in the control group after CPB. Pathological study revealed significantly more severe congestion, alveolar hemorrhage, neutrophil accumulation, and edema, and the number of lung cells expressing HMGB1 increased in the control group. The mRNA expression levels of TNF-α, IL-6, IL-1β, and HMGB1 in the control group were significantly higher than those in other groups. According to Western blot analysis, nuclear factor-kappa B p65 in lung tissue was significantly downregulated in the rTM group. The number of apoptotic cells and the protein of cleaved Caspase-3 were reduced in the rTM group. Conclusions These results suggest that rTM prevents ALI through attenuating inflammation and apoptosis during and after CPB in a rat model.
Recent studies demonstrate that microRNAs show promising potential, including angiogenesis, in therapeutic intervention. MicroRNA-126 (miR-126) is reported to regulate angiogenesis by blocking ...Sprouty-related EVH1 domain-containing protein 1 (SPRED1), an endogenous inhibitor of vascular endothelial cell growth factor. In this study, we investigated the angiogenic effects of the sustained release of miR-126 loaded with poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) in a murine hindlimb ischemia model.
We induced mice hindlimb ischemia through femoral artery excision. We randomly assigned the mice to two groups and performed an intramuscular injection of miR-126-loaded PLGA NPs (miR-126) or scrambled miR-loaded PLGA NPs (control) shortly after induction of ischemia.
The miR-126 expression levels in the ischemic limb at 3 days after treatment were significantly higher in mice treated with miR-126-loaded PLGA NPs than in those with scrambled miR, indicating the fair efficiency of local miR transduction (control vs miR-126: 0.33 ± 0.12 vs 0.74 ± 0.42; P < .05; n = 6). Laser Doppler perfusion imaging revealed that limb blood flow in mice treated with miR-126-loaded PLGA NPs was significantly higher at 14 days after treatment (sham vs control vs miR-126: 0.62 ± 0.09 vs 0.58 ± 0.05 vs 0.72 ± 0.07; P < .001; n = 12). Immunohistochemical analysis indicated that CD31-positive cell density and α-smooth muscle actin-positive vessel density were significantly higher in miR-126-treated mice (control vs miR-126: 0.33 ± 0.12 vs 0.74 ± 0.42; P < .05; n = 6). SPRED1 messenger RNA expression levels were significantly lower in miR-126-treated mice (control vs miR-126: 1.00 ± 0.05 vs 0.81 ± 0.07; P < .05; n = 6). Western blotting indicated that protein levels of pERK/ERK mediated by SPRED1 were significantly higher in miR-126-treated mice (control vs miR-126: 0.29 ± 0.10 vs 0.54 ± 0.21; P < .05; n = 6).
This study suggests that sustained release of miR-126-loaded PLGA NPs might be an effective method in therapeutic angiogenesis for hindlimb ischemia.
Severe clinical limb ischemia resistant to conventional treatments, including medication, percutaneous angioplasty, and bypass surgery, remains a serious health care problem. Therapeutic angiogenesis that induces angiogenesis at the ischemic site could be a novel and effective approach for this severe condition. Here, we developed a therapeutic approach combining microRNA-126 and a drug delivery system using poly lactic-co-glycolic acid nanoparticles, which showed novel therapeutic potential on a mouse hindlimb ischemia model. This therapeutic approach could be a promising new strategy after verification of the safety and long-term therapeutic effects in clinical studies in the future.
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 μL of sustained release form of platelet-poor plasma (PPP), 100 μL of solution form of PRP (PRP-sol), 100 μL 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/mm2 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.
Background:The aim of this study was to assess the long-term outcomes of aortic valve replacement (AVR) with either mechanical or bioprosthetic valves according to age at operation.Methods and ...Results:A total of 1,002 patients (527 mechanical valves and 475 bioprosthetic valves) undergoing first-time AVR were categorized according to age at operation: group Y, age <60 years; group M, age 60–69 years; and group O, age ≥70 years). Outcomes were compared on propensity score analysis (adjusted for 28 variables). Hazard ratio (HR) was calculated using the Cox regression model with adjustment for propensity score with bioprosthetic valve as a reference (HR=1). There were no significant differences in overall mortality between mechanical and bioprosthetic valves for all age groups. Valve-related mortality was significantly higher for mechanical valves in group O (HR, 2.53; P=0.02). Reoperation rate was significantly lower for mechanical valves in group Y (HR, 0.16; P<0.01) and group M (no events for mechanical valves). Although the rate of thromboembolic events was higher in mechanical valves in group Y (no events for tissue valves) and group M (HR, 9.05; P=0.03), there were no significant differences in bleeding events between all age groups.Conclusions:The type of prosthetic valve used in AVR does not significantly influence overall mortality.
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