The vascular endothelium acts as an important component of the vascular system. It is a barrier between the blood and vessel wall. It plays an important role in regulating blood vessel tone, permeability, angiogenesis, and platelet functions. Several studies have shown that amino acids (AA) are key regulators in maintaining vascular homeostasis by modulating endothelial cell (EC) proliferation, migration, survival, and function. This review summarizes the metabolic and signaling pathways of AAs in ECs and discusses the importance of AA homeostasis in the functioning of ECs and vascular homeostasis. It also discusses the challenges in understanding the role of AA in the development of cardiovascular pathophysiology and possible directions for future research.

To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure. 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 (1x10⁶) were seeded into a fibrin/thrombin patch and implanted on the rat infarcted left ventricular anterior wall after myocardial infarction (MI). Echo function was determined at 1- and 6- weeks post-MI. Immunohistochemistry study was performed at week-6 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 left ventricle (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. Transient overexpression of Ang-1 enhanced hiPSC-aCM mitosis and engraftment and increased the reparability potency of hiPSC-aCMs for treatment of MI. Cellular cardiomyoplasty using hiPSC-CMs alone may be insufficient to recover the structure and function of infarcted myocardium in which blood vessels and perfusion are damaged. A combinational approach of myogenesis with vasculogenesis to repopulate CMs with functional neovascular network is necessary to recover LV structure and function post-MI. This is the first report showing evidence that transplantation of hiPSC-CM transiently overexpressing Ang-1 had better cell engraftment and improved heart structure and function post-MI. It highlights that transient genetic modification of hiPSC-CMs with Ang-1 using non-viral vectors offers a better CM based angiomyogenesis therapy for treatment of MI.

The cardiomyocyte undergoes dramatic changes in structure, metabolism, and function from the early fetal stage of hyperplastic cell growth, through birth and the conversion to hypertrophic cell growth, continuing to the adult stage and responding to various forms of stress on the myocardium, often leading to myocardial failure. The fetal cell with incompletely formed sarcomeres and other cellular and extracellular components is actively undergoing mitosis, organelle dispersion, and formation of daughter cells. In the first few days of neonatal life, the heart is able to repair fully from injury, but not after conversion to hypertrophic growth. Structural and metabolic changes occur following conversion to hypertrophic growth which forms a barrier to further cardiomyocyte division, though interstitial components continue dividing to keep pace with cardiac growth. Both intra- and extracellular structural changes occur in the stressed myocardium which together with hemodynamic alterations lead to metabolic and functional alterations of myocardial failure. This review probes some of the questions regarding conditions that regulate normal and pathologic growth of the heart.

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.

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. 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. 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.