Generation of a Human Induced Pluripotent Stem Cell Line Expressing a Magnetic Resonance Imaging Reporter Gene.

The objective of the current study is to develop a new method for tracking transplanted human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) using magnetic resonance imaging (MRI). The CRISPR/dCas9 activation system is employed to overexpress ferritin heavy chain (FHC) in hiPSC-CMs. The mRNA and protein expression of FHC in hiPSC and hiPSC-CMs significantly increased after transfection. Iron chloride does not affect the cell viability in a concentration range from 0 to 2000 µm. hiPSCs overexpressing FHC (hiPSC- FHCOE) and hiPSC-CMs overexpressing FHC (hiPSC-CM-FHCOE) significantly enhanced cellular uptake of iron chloride but with no changes in electrophysiological properties compared to hiPSC-CM-Control. Furthermore, hiPSC-CM-FHCOE presented robust contrast and lower T2* values, signifying their potential as highly effective candidates for cardiac MRI. Next, hiPSC-CM-FHCOE is injected into mouse hearts and after 3 days of transplantation, MR images are obtained. hiPSC-CM-FHCOE cells exhibited clear signals in the hearts with lower T2* and rapid signal decay. Collectively, data from this proof-of-concept study demonstrated that endogenous labeling with FHC in hiPSC-CMs can be a potent strategy for enhancing the accuracy of cardiac MRI. This technology represents a significant step forward in tracking the transplanted hiPSC-CMs in the hearts of live animals.

Linoleic acid-induced ANGPTL4 inhibits C2C12 skeletal muscle differentiation by suppressing Wnt/ß-catenin.

Skeletal muscle differentiation is an essential process in embryonic development as well as regeneration and repair throughout the lifespan. It is well-known that dietary fat intake impacts biological and physiological function in skeletal muscle, however, understanding of the contribution of nutritional factors in skeletal muscle differentiation is limited. Therefore, the objective of the current study was to evaluate the effects of free fatty acids (FFAs) on skeletal muscle differentiation in vitro. We used C2C12 murine myoblasts and treated them with various FFAs, which revealed a unique response of angiopoietin-like protein-4 (ANGPTL4) with linoleic acid (LA) treatment that was associated with reduced differentiation. LA significantly inhibited myotube formation and lowered the protein expression of myogenic regulatory factors, including MyoD and MyoG and increased Pax7 during cell differentiation. Next, recombinant ANGPTL4 protein or siRNA knockdown of ANGPTL4 was employed to examine its role in skeletal muscle differentiation, and we confirmed that ANGPTL4 knockdown at day two and six of differentiation restored myotube formation in the presence of LA. RNA-sequencing analysis revealed that ANGPTL4-mediated inhibition of skeletal muscle differentiation at day two as well as LA at day two or -6 led to a reduction in Wnt/ß-catenin signaling pathways. We confirmed that LA reduced Wnt11 and Axin2 while increasing expression of the Wnt inhibitor, Dkk2. ANGPTL4 knockdown increased ß-catenin protein in the nucleus in response to LA and increased Axin2 and Wnt11 expression. Taken together, these results demonstrate that LA induced ANGPTL4 inhibits C2C12 differentiation by suppressing Wnt/ß-catenin signaling.

Nanoparticle Based Cardiac Specific Drug Delivery.

Heart failure secondary to myocardial injuries is a leading cause of death worldwide. Recently, a growing number of novel therapies have emerged for injured myocardium repairment. However, delivering therapeutic agents specifically to the injured heart remains a significant challenge. Nanoparticles are the most commonly used vehicles for targeted drug delivery. Various nanoparticles have been synthesized to deliver drugs and other therapeutic molecules to the injured heart via passive or active targeting approaches, and their targeting specificity and therapeutic efficacies have been investigated. Here, we summarized nanoparticle-based, cardiac-specific drug delivery systems, their potency for treating heart diseases, and the mechanisms underlying these cardiac-targeting strategies. We also discussed the clinical studies that have employed nanoparticle-based cardiac-specific drug delivery.

A cottonseed oil-enriched diet improves liver and plasma lipid levels in a male mouse model of fatty liver.

A high-fat (HF) diet causes fatty liver, hyperlipidemia, and hypercholesterolemia, and cottonseed oil (CSO) has been shown to improve liver and plasma lipids in human and mouse models. The purpose of this study was to determine the effect of CSO vs. olive oil (OO)-enriched diets on lipid levels in a HF-diet model of fatty liver disease. We placed mice on a HF diet to induce obesity and fatty liver, after which mice were placed on CSO or OO diets, with chow and HF (5.1 kcal/g) groups as control. When CSO- and OO-fed mice were given isocaloric diets with the HF group, there were no differences in body weight, plasma, or hepatic lipids. However, when the CSO and OO diets were reduced in calories (4.0 kcal/g), CSO and OO groups reduced body weight. The CSO group had lower plasma total cholesterol (-56 ± 6%, P < 0.01), free cholesterol (-53 ± 7%, P < 0.01), triglycerides (-61 ± 14%, P < 0.01), and LDL (-42 ± 16%, P = 0.01) vs. HF group whereas the OO diet lowered LDL (-18 ± 12%, P = 0.05) vs. HF. Furthermore, the CSO diet decreased hepatic total cholesterol (-40 ± 12%, P < 0.01), free cholesterol (-23 ± 11%, P = 0.04), and triglycerides (-47 ± 12%, P = 0.02). There were no significant changes in lipogenesis and fatty acid oxidation among the groups. However, the CSO group increased lipid oxidative gene expression in liver and dihydrosterculic acid increased PPARα target genes with in vitro models. Taken together, consuming a reduced calorie diet enriched in CSO reduces liver and plasma lipid profiles in an obese model of fatty liver.

Gene Therapy for Cardiomyocyte Renewal: Cell Cycle, a Potential Therapeutic Target.

Heart disease is the primary cause of death worldwide. Even though extensive research has been done, and many pharmacological and surgical treatments have been introduced to treat heart disease, the mortality rate still remains high. Gene therapy is widely used to understand molecular mechanisms of myocardial infarction and to treat cardiomyocyte loss. It was reported that adult cardiomyocytes proliferate at a very low rate; thus, targeting their proliferation has become a new regenerative therapeutic approach. Currently, re-activating cardiomyocyte proliferation appears to be one of the most promising methods to promote adult cardiomyocyte renewal. In this article, we highlight gene therapeutic targets of cell proliferation presently being pursued to re-activate the cell cycle of cardiomyocytes, including cell cycle regulators, transcription factors, microRNAs, signal transduction, and other contributing factors. We also summarize gene delivery vectors that have been used in cardiac research and major challenges to be overcome in the translation to the clinical approach and future directions.

A Review of free fatty acid-induced cell signaling, angiopoietin-like protein 4, and skeletal muscle differentiation.

Postnatal skeletal muscle differentiation from quiescent satellite cells is a highly regulated process, although our understanding of the contribution of nutritional factors in myogenesis is limited. Free fatty acids (FFAs) are known to cause detrimental effects to differentiated skeletal muscle cells by increasing oxidative stress which leads to muscle wasting and insulin resistance in skeletal muscle. In addition, FFAs are thought to act as inhibitors of skeletal muscle differentiation. However, the precise molecular mechanisms underlying the effects of FFAs on skeletal muscle differentiation remains to be elucidated. There is a clear relationship between dietary FFAs and their ability to suppress myogenesis and we propose the hypothesis that the FFA-mediated increase in angiopoietin-like protein 4 (ANGPTL4) may play a role in the inhibition of differentiation. This review discusses the role of FFAs in skeletal muscle differentiation to-date and proposes potential mechanisms of FFA-induced ANGPTL4 mediated inhibition of skeletal muscle differentiation.

Free Fatty Acid-Induced Peptide YY Expression Is Dependent on TG Synthesis Rate and Xbp1 Splicing.

Gut-derived satiety hormones provide negative feedback to suppress food intake and maintain metabolic function in peripheral tissues. Despite the wealth of knowledge of the systemic effects of these hormones, very little is known concerning the mechanisms by which nutrients, such as dietary fats, can promote the expression of genes involved in L-cell hormone production. We have tested the role of various dietary fats and found that after hydrolysis into free fatty acids (FFA's), there is a differential response in the extent to which they induce PYY gene and protein production. The effect of FFA's also seems to relate to triglyceride (TG) re-esterification rate, with MUFA re-esterifying faster with lower PYY production. We have also found that there are differences in potency of FFA's based on their desaturation patterns in vitro. The potency effect of FFA's is influenced by the rate of TG re-esterification, such that the longer FFA's are in contact with L-cells, the more PYY they produce. We found that chronic consumption of high-fat diets enables the small intestine to re-esterify FFA's into TG faster and earlier which resulted in a blunted postprandial PYY response. Lastly, we found that FFA's induce X-box-binding protein-1 activation (Xbp1s) in L-cells and that adenoviral delivery of Xbp1s was sufficient to induce PYY gene expression. Taken together, the present work indicates that dietary fat can induce satiety, in part, prior to re-esterification. Chronic high-fat diet consumption increases the rate of re-esterification which diminishes satiety and may lead to increased food intake. Targeting intestinal TG synthesis may prove beneficial in restoring obesity-associated reductions in postprandial satiety.

Angiopoietin-1 protects 3T3-L1 preadipocytes from saturated fatty acid-induced cell death.

Cross talk between endothelial cells and adipocytes is vital to adipocyte functions, but little is known about the mechanisms or factors controlling the process. Angiogenesis is a critical component linking the endothelium to healthy adipogenesis, yet it is not known if or how it is involved in adipocyte physiology. Therefore, the purpose of this study was to determine the effect of angiopoietin-1 (Ang-1) and -2 (Ang-2) as well as their receptor, Tie-2, on adipocyte physiology. 3T3-L1 pre- and mature adipocytes were found to express Ang-1, Ang-2, and Tie-2, which decrease upon polyunsaturated fatty acid treatment. Furthermore, 3T3-L1 cells treated with recombinant Ang-1 or Ang-2 increased expression of the antiapoptotic gene Bcl-x and decreased expression of the proapoptotic gene Casp-8. Next, preadipocytes were treated with saturated fatty acids (SFAs) to induce cell stress. SFA-mediated splicing of X-box-binding protein-1 was reduced by co-treatment with Ang-1, and cell viability was improved in the presence of SFAs + Ang-1. Taken together, these results indicate that Ang-1 may protect preadipocytes from SFA-induced apoptosis and endoplasmic reticulum stress.

Pharmacokinetics and Plasma Cellular Antioxidative Effects of Flavanols After Oral Intake of Green Tea Formulated with Vitamin C and Xylitol in Healthy Subjects.

This study aimed to test whether green tea formulated with vitamin C and xylitol (GTVX) could improve absorption of flavanols and total antioxidant activity (TAC) of plasma compared with green tea only (GT) in healthy subjects. The total radical-trapping antioxidant parameter method was used to measure the TAC of plasma. Cmax, Tmax, and area under the curve (AUC) of flavanols in plasma after consumption of GTVX were 5980.58 µg/mL, 2.14 h, and 18,915.56 h·µg/mL, respectively, indicating that GTVX showed significantly higher AUC than GT (13,855.43 µg/mL). The peak TACs occurred at 3 and 0.5 h after intake of GT and GTVX, respectively. The TAC of plasma was found to be significantly higher in GTVX than in GT at each time point. This study suggests that formulating green tea with vitamin C and xylitol could increase the absorption of flavanols in green tea, enhancing cellular antioxidative effects.

Combinational enhancing effects of formulation and encapsulation on digestive stability and intestinal transport of green tea catechins.

The hypothesis was that green tea catechins (GTCs) formulated with vitamin C and xylitol followed by enteric coating with hydroxypropyl methyl cellulose phthalate (HPMCP) or encapsulated into γ-cyclodextrin (γ-CD) could enhance intestinal absorption of GTCs. Surface morphology and size obtained by SEM were different. Digestive stability of GTCs encapsulated into γ-CD or coated with HPMCP was enhanced up to 65.56% or 57.63%, respectively. When GTCs were formulated, the digestive stability was greater than the one not formulated. Formulated GTCs followed by encapsulation into γ-CD significantly increased intestinal transport. Absorption of GTCs was 2.8%, 9.64%, 11.97%, 8.41% and 14.36% for only GTCs, GTCs encapsulated into γ-CD, formulated GTCs encapsulated into γ-CD, GTCs coated with HPMCP and formulated GTCs coated with HPMCP, respectively. This study suggests that GTCs, formulated with vitamin C and xylitol followed by γ-CD encapsulation or HPMCP enteric coating, provide combinational effect to increase bioavailability of GTCs.

Bioefficacy of Graviola leaf extracts in scavenging free radicals and upregulating antioxidant genes.

The aims of this study were to determine bioactive components of Graviola leaf extracts and to examine the radical scavenging capacity, gene expression and transcription factors of antioxidant enzymes. Rutin, kaempferol-rutinoside, and vitamin U were identified from the steaming and 50% EtOH extracts of Graviola leaves. Graviola leaf extracts effectively scavenged peroxy and nitrogen radicals. 50% EtOH of Graviola leaves provided a 1-2.9 times higher trolox equivalent than the steaming extract. It also had a higher VCEAC. Graviola leaf extracts reduced the generation of reactive oxygen species (ROS) induced by H2O2 in a dose-dependent manner. The 50% EtOH extract of Graviola leaves upregulated SOD1 and Nrf2, but catalase and HMOX1 were not altered by the 50% EtOH extract of Graviola leaves.