Thymosin beta-4 improves endothelial function and reparative potency of diabetic endothelial cells differentiated from patient induced pluripotent stem cells.

BACKGROUND: Prior studies show that signature phenotypes of diabetic human induced pluripotent stem cells derived endothelial cells (dia-hiPSC-ECs) are disrupted glycine homeostasis, increased senescence, impaired mitochondrial function and angiogenic potential as compared with healthy hiPSC-ECs. In the current study, we aimed to assess the role of thymosin ß-4 (Tb-4) on endothelial function using dia-hiPSC-ECs as disease model of endothelial dysfunction. METHODS AND RESULTS: Using dia-hiPSC-ECs as models of endothelial dysfunction, we determined the effect of Tb-4 on cell proliferation, senescence, cyto-protection, protein expression of intercellular adhesion molecule-1 (ICAM-1), secretion of endothelin-1 and MMP-1, mitochondrial membrane potential, and cyto-protection in vitro and angiogenic potential for treatment of ischemic limb disease in a mouse model of type 2 diabetes mellitus (T2DM) in vivo. We found that 600 ng/mL Tb4 significantly up-regulated AKT activity and Bcl-XL protein expression, enhanced dia-hiPSC-EC viability and proliferation, limited senescence, reduced endothelin-1 and MMP-1 secretion, and improved reparative potency of dia-hiPSC-ECs for treatment of ischemic limb disease in mice with T2DM. However, Tb4 had no effect on improving mitochondrial membrane potential and glycine homeostasis and reducing intercellular adhesion molecule-1 protein expression in dia-hiPSC-ECs. CONCLUSIONS: Tb-4 improves endothelial dysfunction through enhancing hiPSC-EC viability, reducing senescence and endothelin-1 production, and improves angiogenic potency in diabetes.

A diastolic dysfunction model in non-human primates with transverse aortic constriction.

Transverse aortic constriction (TAC) has been widely used to study cardiac hypertrophy, fibrosis, diastolic dysfunction, and heart failure in rodents. Few studies have been reported in preclinical animal models. The similar physiology and anatomy between non-human primates (NHPs) and humans make NHPs valuable models for disease modeling and testing of drugs and devices. In the current study, we aimed to establish a TAC model in NHPs and characterize the structural and functional profiles of the heart after TAC. A non-absorbable suture was placed around the aorta between the brachiocephalic artery and left common carotid artery to create TAC. NHPs were divided into 2 groups according to pressure gradient (PG): the Mild Group (PG=31.01 ± 12.40 mmHg, n=3) and the Moderate Group (PG=53.00 ± 9.37 mmHg, n=4). At 4 weeks after TAC, animals in both TAC groups developed cardiac hypertrophy: enlarged myocytes and increased wall thickness of the left ventricular (LV) anterior wall. Although both TAC groups had normal systolic function that was similar to a Sham Group, the Moderate Group showed diastolic dysfunction that was associated with more severe cardiac fibrosis, as evidenced by a reduced A wave velocity, large E wave velocity/A wave velocity ratio, and short isovolumic relaxation time corrected by heart rate. Furthermore, no LV arrhythmia was observed in either animal group after TAC. A diastolic dysfunction model with cardiac hypertrophy and fibrosis was successfully developed in NHPs.

Angiopoietin-1 enhanced myocyte mitosis, engraftment, and the reparability of hiPSC-CMs for treatment of myocardial infarction.

AIMS: To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure. METHODS AND RESULTS: Atrial hiPSC-CMs (hiPSC-aCMs) were differentiated from hiPSCs and purified by lactic acid and were transfected with Ang-1 (Ang-1-hiPSC-aCMs) plasmid using lipoSTEM. Ang-1 gene transfection efficiency was characterized in vitro. Gene transfected CMs (1×106) were seeded into a fibrin/thrombin patch and implanted on the rat-infarcted left ventricular (LV) anterior wall after myocardial infarction (MI). Echo function was determined at 1- and 6 weeks post-MI. Immunohistochemistry study was performed at 6 weeks post-MI. Ang-1 (20 and 40 ng/mL) protected hiPSC-aCMs from hypoxia through up-regulating pERK1/2 and inhibiting Bax protein expressions. Ang-1-hiPSC-aCMs transiently secreted Ang-1 protein up to 14 days, with peak level on day-2 post-transfection (24.39 ± 13.02 ng/mL) in vitro. Animal study showed that transplantation of Ang-1-hiPSC-aCM seeded patch more effectively limited rat heart apoptosis at 1 day post-MI as compared with LipoSTEM-Ang-1 or hiPSC-aCMs transplantation. Ang-1-hiPSC-aCMs transplantation induced host (rat) and donor (human) CM mitosis and arteriole formation, improved cell engraftment rate, more effectively limited LV dilation (EDV = 460.7 ± 96.1 µL and ESV = 219.8 ± 72.9 µL) and improved LV global pump function (EF = 53.1 ± 9%) as compared with the MI (EDV = 570.9 ± 91.8 µL, P = 0.033; ESV = 331.6 ± 71.2 µL, P = 0.011; EF = 42.3 ± 4.1%, P = 0.02) or the LipoSTEM-Ang-1 injected (EDV = 491.4 ± 100.4 µL, P = 0.854; ESV = 280.9 ± 71.5 µL, P = 0.287; EF = 43.2 ± 4.6, P = 0.039) or hiPSC-CM transplanted (EDV = 547.9 ± 55.5 µL, P = 0.095; ESV = 300.2 ± 88.4 µL, P = 0.075; EF = 46 ± 10.9%, P = 0.166) animal groups at 6 weeks post-MI and treatment. CONCLUSION: Transient over-expression of Ang-1 enhanced hiPSC-aCM mitosis and engraftment and increased the reparability potency of hiPSC-aCMs for treatment of MI.

Dexamethasone inhibits regeneration and causes ventricular aneurysm in the neonatal porcine heart after myocardial infarction.

AIMS: Recently, we demonstrated that the hearts of neonatal pigs (2-day old) have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after postnatal day 3. However, it is unknown if corticosteroid, a broad anti-inflammatory agent, will abrogate the regenerative capacity in the hearts of neonatal pigs. The aim of the current study is to evaluate the effect Dexamethasone (Dex), a broad anti-inflammatory agent, on heart regeneration, structure, and function of the neonatal pigs' post-myocardial infarction (MI). METHODS AND RESULTS: Dex (0.2 mg/kg/day) was injected intramuscularly into the neonatal pig (age: 2 days postnatal) during the first week post-MI. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance (CMR) imaging. Bromodeoxyuridine (BrdU) pulse-chase labeling, histology, immunohistochemistry, and flow cytometry were performed to determine inflammatory cell infiltration, CM cytokinesis, and myocardial fibrosis. Dex injection during the first-week suppressed acute inflammation post-MI in the pig hearts. It inhibited BrdU incorporation to pig CMs and CM cytokinesis via inhibiting aurora-B protein expression which was associated with mature scar formation and thinned walls at the infarct site. CMR imaging showed Dex caused left ventricular aneurysm and poor ejection fraction. CONCLUSIONS: Dex inhibited CM cytokinesis and functional recovery and caused ventricular aneurysm in the hearts of 2-day old pigs post-MI.

Non-viral vector based gene transfection with human induced pluripotent stem cells derived cardiomyocytes.

Non-viral transfection of mammalian cardiomyocytes (CMs) is challenging. The current study aims to characterize and determine the non-viral vector based gene transfection efficiency with human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs differentiated from PCBC hiPSCs were used as a cell model to be transfected with plasmids carrying green fluorescence protein (pGFP) using polyethylenimine (PEI), including Transporter 5 Transfection Reagent (TR5) and PEI25, and liposome, including lipofectamine-2000 (Lipo2K), lipofectamine-3000 (Lipo3K), and Lipofectamine STEM (LipoSTEM). The gene transfection efficiency and cell viability were quantified by flow cytometry. We found that the highest gene transfection efficiency in hiPSC-CMs on day 14 of contraction can be achieved by LipoSTEM which was about 32.5 ± 6.7%. However, it also cuased poor cell viability (60.1 ± 4.5%). Furthermore, a prolonged culture of (transfection on day 23 of contraction) hiPSC-CMs not only improved gene transfection (54.5 ± 8.9%), but also enhanced cell viability (74 ± 4.9%) by LipoSTEM. Based on this optimized gene transfection condition, the highest gene transfection efficiency was 55.6 ± 7.8% or 34.1 ± 4%, respectively, for P1C1 or DP3 hiPSC line that was derived from healthy donor (P1C1) or patient with diabetes (DP3). The cell viability was 80.8 ± 5.2% or 92.9 ± 2.24%, respectively, for P1C1 or DP3. LipoSTEM is a better non-viral vector for gene transfection of hiPSC-CMs. The highest pGFP gene transfection efficiency can reach >50% for normal hiPSC-CMs or >30% for diabetic hiPSC-CMs.

The prostaglandin H2 analog U-46619 improves the differentiation efficiency of human induced pluripotent stem cells into endothelial cells by activating both p38MAPK and ERK1/2 signaling pathways.

BACKGROUND: We have shown that the differentiation of human-induced pluripotent stem cells (hiPSCs) into endothelial cells (ECs) is more efficient when performed with a 3-dimensional (3D) scaffold of biomaterial than in monolayers. The current study aims to further increase hiPSC-EC differentiation efficiency by deciphering the signaling pathways in 3D scaffolds. METHODS AND RESULTS: We modified our 3D protocol by using U-46619 to upregulate both p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, which increased the differentiation efficiency (as measured by CD31 expression) to as high as 89% in two established hiPSC lines. The differentiated cells expressed arteriovenous, but not lymphatic, markers; formed tubular structures and EC lumen in vitro; had significantly shorter population-doubling times than monolayer-differentiated hiPSC-ECs; and restored perfusion and vascularity in a murine hind limb ischemia model. The differentiation efficiency was also > 85% in three hiPSC lines that had been derived from patients with diseases or disease symptoms that have been linked to endothelial dysfunction. CONCLUSIONS: These observations demonstrate that activating both p38MAPK and ERK1/2 signaling pathways with U-46619 improves the efficiency of arteriovenous hiPSC-EC differentiation and produces cells with greater proliferative capacity.

Truncations of the titin Z-disc predispose to a heart failure with preserved ejection phenotype in the context of pressure overload.

Titin (TTN) Truncating variants (TTNtv) in the A-band of TTN predispose the mouse heart to systolic dysfunction when subjected to pressure-loading. However, the effects of TTNtv of the Z-disc are largely unexplored. A rat model of pressure-loaded heart is developed by trans-aortic constriction (TAC). Rats with TTNtv of the Z-disc were randomly assigned to TAC (Z-TAC) or sham-surgery (Z-Sham) and wildtype (WT) littermates served as controls (WT-TAC or WT-Sham). Left ventricular (LV) function was assessed by echocardiography. Pressure volume (PV) loops, histology and molecular profiling were performed eight months after surgery. Pressure-load by TAC increased LV mass in all cases when compared with Sham animals. Notably, systolic function was preserved in TAC animals throughout the study period, which was confirmed by terminal PV loops. Diastolic function was impaired in Z-disc TTNtv rats at baseline as compared to WT and became impaired further after TAC (dp/dtmin, mmHg/s): Z-TAC = -3435±763, WT-TAC = -6497±1299 (p<0.01). Z-TAC animals had greater cardiac fibrosis, with elevated collagen content and decreased vascular density as compared to WT-TAC animals associated with enhanced apoptosis of myocyte and non-myocyte populations. In the context of pressure overload, Z-disc TTNtv is associated with cardiac fibrosis, diastolic dysfunction, and capillary rarefaction in the absence of overt systolic dysfunction.