Heterocyclic Amines Disrupt Lipid Homeostasis in Cryopreserved Human Hepatocytes.

Metabolic dysfunction associated-steatotic liver disease (MASLD)/metabolic dysfunction-associated steatohepatitis (MASH) is the liver manifestation of metabolic syndrome, which is characterized by insulin resistance, hyperglycemia, hypertension, dyslipidemia, and/or obesity. Environmental pollutant exposure has been recently identified as a risk factor for developing MASH. Heterocyclic amines (HCAs) are mutagens generated when cooking meat at high temperatures or until well-done. Recent epidemiological studies reported that dietary HCA exposure may be linked to insulin resistance and type II diabetes, and we recently reported that HCAs induce insulin resistance and glucose production in human hepatocytes. However, no previous studies have examined the effects of HCAs on hepatic lipid homeostasis. In the present study, we assessed the effects of two common HCAs, MeIQx (2-amino-3, 8-dimethylimidazo [4, 5-f] quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4, 5-b] pyridine), on lipid homeostasis in cryopreserved human hepatocytes. Exposure to a single concentration of 25 µM MeIQx or PhIP in human hepatocytes led to dysregulation of lipid homeostasis, typified by significant increases in lipid droplets and triglycerides. PhIP significantly increased expression of lipid droplet-associated genes, PNPLA3 and HSD17B13, and both HCAs significantly increased PLIN2. Exposure to MeIQx or PhIP also significantly increased expression of several key genes involved in lipid synthesis, transport and metabolism, including FASN, DGAT2, CPT1A, SCD, and CD36. Furthermore, both MeIQx and PhIP significantly increased intracellular cholesterol and decreased expression of PON1 which is involved in cholesterol efflux. Taken together, these results suggest that HCAs dysregulate lipid production, metabolism, and storage. The current study demonstrates, for the first time, that HCA exposure may lead to fat accumulation in hepatocytes, which may contribute to hepatic insulin resistance and MASH.

The Unfolded Protein Response and Its Implications for Novel Therapeutic Strategies in Inflammatory Bowel Disease.

The endoplasmic reticulum (ER) is a multifunctional organelle playing a vital role in maintaining cell homeostasis, and disruptions to its functions can have detrimental effects on cells. Dysregulated ER stress and the unfolded protein response (UPR) have been linked to various human diseases. For example, ER stress and the activation of the UPR signaling pathways in intestinal epithelial cells can either exacerbate or alleviate the severity of inflammatory bowel disease (IBD), contingent on the degree and conditions of activation. Our recent studies have shown that EPICERTIN, a recombinant variant of the cholera toxin B subunit containing an ER retention motif, can induce a protective UPR in colon epithelial cells, subsequently promoting epithelial restitution and mucosal healing in IBD models. These findings support the idea that compounds modulating UPR may be promising pharmaceutical candidates for the treatment of the disease. In this review, we summarize our current understanding of the ER stress and UPR in IBD, focusing on their roles in maintaining cell homeostasis, dysregulation, and disease pathogenesis. Additionally, we discuss therapeutic strategies that promote the cytoprotection of colon epithelial cells and reduce inflammation via pharmacological manipulation of the UPR.

Induction of glucose production by heterocyclic amines is dependent on N-acetyltransferase 2 genetic polymorphism in cryopreserved human hepatocytes.

Heterocyclic amines (HCAs) are mutagenic compounds found in cooked meat. Recent epidemiological studies reported significant associations between dietary HCA exposure and insulin resistance and type II diabetes, and we recently reported that HCAs induce insulin resistance and glucose production in human hepatocytes. It is well known that HCAs require hepatic bioactivation by cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2). NAT2 expresses a well-defined genetic polymorphism in humans that, depending on the combination of NAT2 alleles, correlates to rapid, intermediate, or slow acetylator phenotype that exhibits differential metabolism of aromatic amines and HCAs. No previous studies have examined the role of NAT2 genetic polymorphism in the context of HCA-mediated induction of glucose production. In the present study, we assessed the effect of three HCAs commonly found in cooked meat (2-amino-3,4-dimethylimidazo[4,5-f]quinoline [MeIQ], 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [PhIP]) on glucose production in cryopreserved human hepatocytes with slow, intermediate, or rapid NAT2 acetylator phenotype. HCA treatment did not affect glucose production in slow NAT2 acetylator hepatocytes, while a slight increase in glucose production was observed in intermediate NAT2 acetylators treated with MeIQ or MeIQx. However, significant increases in glucose production were observed in rapid NAT2 acetylators following each HCA. The current findings suggest that individuals who are rapid NAT2 acetylators may be at a greater risk of developing hyperglycemia and insulin resistance following dietary exposure to HCAs.

N -acetyltransferase 2 haplotype modifies risks for both dyslipidemia and urinary bladder cancer.

A novel haplotype in N -acetyltransferase 2 ( NAT2 ) composed of seven non-coding variants (rs1495741, rs4921913, rs4921914, rs4921915, rs146812806, rs35246381, and rs35570672) has been linked to dyslipidemia by multiple, independent genome-wide association studies. The haplotype is located approximately 14 kb downstream of NAT2-coding region (ch8:18,272,377-18,272,881; GRCh38/hg38) and represents a non-coding, intergenic haplotype. Interestingly, the same dyslipidemia NAT2 haplotype is also linked to urinary bladder cancer risk. Dyslipidemia risk alleles are associated with rapid acetylator phenotype, whereas bladder cancer risk alleles are associated with slow acetylator, suggesting that the level of systemic NAT2 activity modifies the risk of these pathologies. We speculate that rs1495741 (and its associated haplotype) belongs to a distal regulatory element of human NAT2 gene (e.g., enhancer or silencer), and the genetic variation at the newly discovered haplotype results in a differential level of NAT2 gene expression. Understanding how this NAT2 haplotype contributes to not only urinary bladder cancer but also to dyslipidemia will ultimately help devise strategies to identify and protect susceptible individuals.

Heterocyclic amines reduce insulin-induced AKT phosphorylation and induce gluconeogenic gene expression in human hepatocytes.

Heterocyclic amines (HCAs) are well-known for their mutagenic properties. One of the major routes of human exposure is through consumption of cooked meat, as certain cooking methods favor formation of HCAs. Recent epidemiological studies reported significant associations between dietary HCA exposure and insulin resistance and type II diabetes. However, no previous studies have examined if HCAs, independent of meat consumption, contributes to pathogenesis of insulin resistance or metabolic disease. In the present study, we have assessed the effect of three HCAs commonly found in cooked meat (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [MeIQ], 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [PhIP]) on insulin signaling and glucose production. HepG2 or cryopreserved human hepatocytes were treated with 0-50 µM of MeIQ, MeIQx, or PhIP for 3 days. Treatment of HepG2 cells and hepatocytes with MeIQ and MeIQx resulted in a significant reduction in insulin-induced AKT phosphorylation, suggesting that HCA exposure decreases hepatic insulin signaling. HCA treatment also led to significant increases in expression of gluconeogenic genes, G6PC and PCK1, in both HepG2 and cryopreserved human hepatocytes. Additionally, the level of phosphorylated FOXO1, a transcriptional regulator of gluconeogenesis, was significantly reduced by HCA treatment in hepatocytes. Importantly, HCA treatment of human hepatocytes led to increases in extracellular glucose level in the presence of gluconeogenic substrates, suggesting that HCAs induce hepatic glucose production. The current findings suggest that HCAs induce insulin resistance and promote hepatic glucose production in human hepatocytes. This implicates that exposure to HCAs may lead to the development of type II diabetes or metabolic syndrome.

Non-coding and intergenic genetic variants of human arylamine N-acetyltransferase 2 (NAT2) gene are associated with differential plasma lipid and cholesterol levels and cardiometabolic disorders.

Arylamine N-acetyltransferase 2 (NAT2) is a phase II metabolic enzyme, best known for metabolism of aromatic amines and hydrazines. Genetic variants occurring in the NAT2 coding region have been well-defined and are known to affect the enzyme activity or protein stability. Individuals can be categorized into rapid, intermediate, and slow acetylator phenotypes that significantly alter their ability to metabolize arylamines, including drugs (e.g., isoniazid) and carcinogens (e.g., 4-aminobiphenyl). However, functional studies on non-coding or intergenic variants of NAT2 are lacking. Multiple, independent genome wide association studies (GWAS) have reported that non-coding or intergenic variants of NAT2 are associated with elevated plasma lipid and cholesterol levels, as well as cardiometabolic disorders, suggesting a novel cellular role of NAT2 in lipid and cholesterol homeostasis. The current review highlights and summarizes GWAS reports that are relevant to this association. We also present a new finding that seven, non-coding, intergenic NAT2 variants (i.e., rs4921913, rs4921914, rs4921915, rs146812806, rs35246381, rs35570672, and rs1495741), which have been associated with plasma lipid and cholesterol levels, are in linkage disequilibrium with one another, and thus form a novel haplotype. The dyslipidemia risk alleles of non-coding NAT2 variants are associated with rapid NAT2 acetylator phenotype, suggesting that differential systemic NAT2 activity might be a risk factor for developing dyslipidemia. The current review also discusses the findings of recent reports that are supportive of the role of NAT2 in lipid or cholesterol synthesis and transport. In summary, we review data suggesting that human NAT2 is a novel genetic factor that influences plasma lipid and cholesterol levels and alters the risk of cardiometabolic disorders. The proposed novel role of NAT2 merits further investigations.

Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1), a phase II metabolic enzyme, is frequently upregulated in breast cancer. Inhibition or depletion of NAT1 leads to growth retardation in breast cancer cells in vitro and in vivo. A previous metabolomics study of MDA-MB-231 breast cancer cells suggests that NAT1 deletion leads to a defect in de novo pyrimidine biosynthesis. In the present study, we observed that NAT1 deletion results in upregulation of cytidine deaminase (CDA), which is involved in the pyrimidine salvage pathway, in multiple breast cancer cell lines (MDA-MB-231, MCF-7 and ZR-75-1). We hypothesized that NAT1 KO MDA-MB-231 cells show differential sensitivity to drugs that either inhibit cellular pyrimidine homeostasis or are metabolized by CDA. METHODS: The cells were treated with (1) inhibitors of dihydroorotate dehydrogenase or CDA (e.g., teriflunomide and tetrahydrouridine); (2) pyrimidine/nucleoside analogs (e.g., gemcitabine and 5-azacytidine); and (3) naturally occurring, modified cytidines (e.g., 5-formyl-2'-deoxycytidine; 5fdC). RESULTS: Although NAT1 KO cells failed to show differential sensitivity to nucleoside analogs that are metabolized by CDA, they were markedly more sensitive to 5fdC which induces DNA damage in the presence of high CDA activity. Co-treatment with 5fdC and a CDA inhibitor, tetrahydrouridine, abrogated the increase in 5fdC cytotoxicity in NAT1 KO cells, suggesting that the increased sensitivity of NAT1 KO cells to 5fdC is dependent on their increased CDA activity. CONCLUSIONS: The present findings suggest a novel therapeutic strategy to treat breast cancer with elevated NAT1 expression. For instance, NAT1 inhibition may be combined with cytotoxic nucleosides (e.g., 5fdC) for breast cancer treatment.

Dataset for proteomic analysis of arylamine N-acetyltransferase 1 knockout MDA-MB-231 breast cancer cells.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. An unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines were performed. Among 4,890 proteins identified, 737 and 651 proteins were found significantly (p < 0.01) upregulated and downregulated, respectively, in NAT1 KO cells, compared to the parental cells. Each set of proteins was analyzed to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in the set. Among the proteins upregulated in NAT1 KO cells, processes associated with MHC major histocompatibility complex I-mediated antigen presentation were significantly enriched. Multiple processes involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells. The current dataset contains additional representations of the biological processes and components that are differentially enriched in NAT1 KO MDA-MB-231 cells and will serve as a basis for generating novel hypotheses regarding the role of NAT1 in breast cancer. Data are available via ProteomeXchange with identifier PXD035953.

Proteomic analysis of arylamine N-acetyltransferase 1 knockout breast cancer cells: Implications in immune evasion and mitochondrial biogenesis.

Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1's role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation.

Transcriptional Regulation of Human Arylamine N-Acetyltransferase 2 Gene by Glucose and Insulin in Liver Cancer Cell Lines.

Arylamine N-acetyltransferase 2 (NAT2) is well-known for its role in phase II metabolism of xenobiotics and drugs. More recently, genome wide association studies and murine models implicated NAT2 in regulation of insulin sensitivity and plasma lipid levels. However, the mechanism remains unknown. Transcript levels of human NAT2 varied dynamically in HepG2 (hepatocellular) cells, depending on the nutrient status of the culture media. Culturing the cells in the presence of glucose induced NAT2 mRNA expression as well as its N-acetyltransferase activity significantly. In addition, insulin or acetate treatment also significantly induced NAT2 mRNA. We examined and compared the glucose- and acetate-dependent changes in NAT2 expression to those of genes involved in glucose and lipid metabolism, including FABP1, CPT1A, ACACA, SCD, CD36, FASN, ACLY, G6PC, and PCK1. Genes that are involved in fatty acid transport and lipogenesis, such as FABP1 and CD36, shared a similar pattern of expression with NAT2. In silico analysis of genes co-expressed with NAT2 revealed an enrichment of biological processes involved in lipid and cholesterol biosynthesis and transport. Among these, A1CF (APOBEC1 complementation factor) showed the highest correlation with NAT2 in terms of its expression in normal human tissues. The current study shows, for the first time, that human NAT2 is transcriptionally regulated by glucose and insulin in liver cancer cell lines and that the gene expression pattern of NAT2 is similar to that of genes involved in lipid metabolism and transport.

Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

Acetylator Genotype-Dependent Dyslipidemia in Rats Congenic for N-Acetyltransferase 2.

Recent reports suggest that arylamine N-acetyltransferases (NAT1 and/or NAT2) serve important roles in regulation of energy utility and insulin sensitivity. We investigated the interaction between diet (control vs. high-fat diet) and acetylator phenotype (rapid vs. slow) using previously established congenic rat lines (in F344 background) that exhibit rapid or slow Nat2 (orthologous to human NAT1) acetylator genotypes. Male and female rats of each genotype were fed control or high-fat (Western-style) diet for 26 weeks. We then examined diet- and acetylator genotype-dependent changes in body and liver weights, systemic glucose tolerance, insulin sensitivity, and plasma lipid profile. Male and female rats on the high fat diet weighed approximately 10% more than rats on the control diet and the percentage liver to body weight was consistently higher in rapid than slow acetylator rats. Rapid acetylator rats were more prone to develop dyslipidemia overall (i.e., higher triglyceride; higher LDL; and lower HDL), compared to slow acetylator rats. Total cholesterol (TC)-to-HDL ratios were significantly higher and HDL-to-LDL ratios were significantly lower in rapid acetylator rats. Our data suggest that rats with rapid systemic Nat2 (NAT1 in humans) genotype exhibited higher dyslipidemia conferring risk for metabolic syndrome and cardiovascular dysfunction.

CRISPR/Cas9 knockout of human arylamine N-acetyltransferase 1 in MDA-MB-231 breast cancer cells suggests a role in cellular metabolism.

Human arylamine N-acetyltransferase 1 (NAT1), present in all tissues, is classically described as a phase-II xenobiotic metabolizing enzyme but can also catalyze the hydrolysis of acetyl-Coenzyme A (acetyl-CoA) in the absence of an arylamine substrate using folate as a cofactor. NAT1 activity varies inter-individually and has been shown to be overexpressed in estrogen receptor-positive (ER+) breast cancers. NAT1 has also been implicated in breast cancer progression however the exact role of NAT1 remains unknown. The objective of this study was to evaluate the effect of varying levels of NAT1 N-acetylation activity in MDA-MB-231 breast cancer cells on global cellular metabolism and to probe for unknown endogenous NAT1 substrates. Global, untargeted metabolomics was conducted via ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on MDA-MB-231 breast cancer cell lines constructed with siRNA and CRISPR/Cas9 technologies to vary only in NAT1 N-acetylation activity. Many metabolites were differentially abundant in NAT1-modified cell lines compared to the Scrambled parental cell line. N-acetylasparagine and N-acetylputrescine abundances were strongly positively correlated (r = 0.986 and r = 0.944, respectively) with NAT1 N-acetylation activity whereas saccharopine abundance was strongly inversely correlated (r = -0.876). Two of the most striking observations were a reduction in de novo pyrimidine biosynthesis and defective ß-oxidation of fatty acids in the absence of NAT1. We have shown that NAT1 expression differentially affects cellular metabolism dependent on the level of expression. Our results support the hypothesis that NAT1 is not just a xenobiotic metabolizing enzyme and may have a role in endogenous cellular metabolism.

N-Acetyltransferase 1 Knockout Elevates Acetyl Coenzyme A Levels and Reduces Anchorage-Independent Growth in Human Breast Cancer Cell Lines.

Elevated expression of N-acetyltransferase 1 (NAT1) is associated with invasive and lobular breast carcinomas as well as with bone metastasis following an epithelial-to-mesenchymal transition. We investigated the effect of NAT1 gene deletion in three different human breast cancer cell lines, MDA-MB-231, MCF-7, and ZR-75-1. Human NAT1 was knocked out using CRISPR/Cas9 technology and two different guide RNAs. None of the NAT1 knockout (KO) cell lines exhibited detectable NAT1 activity when measured using its selective substrate p-aminobenzoic acid (PABA). Endogenous acetyl coenzyme A levels (cofactor for acetylation pathways) in NAT1 KO cell lines were significantly elevated in the MDA-MB-231 (p < 0.001) and MCF-7 (p=0.0127) but not the ZR-75-1 (p > 0.05). Although the effects of NAT1 KO on cell-doubling time were inconsistent across the three breast cancer cell lines, the ability of the NAT1 KO cell lines to form anchorage-independent colonies in soft agar was dramatically and consistently reduced in each of the breast cancer cell lines. The NAT1 KO clones for MDA-MB-231, MCF-7, and ZR-75-1 had a reduction greater than 20-, 6-, and 7- folds in anchorage-independent cell growth, respectively, compared to their parental cell lines (p < 0.0001, p < 0.0001, and p < 0.05, respectively). The results indicate that NAT1 may be an important regulator of cellular acetyl coenzyme A levels and strongly suggest that elevated NAT1 expression in breast cancers contribute to their anchorage-independent growth properties and ultimately metastatic potential.

Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

Repeated doses of cardiac mesenchymal cells are therapeutically superior to a single dose in mice with old myocardial infarction.

We have recently demonstrated that repeated administrations of c-kitPOS cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7% vs. 32.7 ± 0.6% in the single-dose group (P < 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit POS CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.

The Epigenetic Regulator HDAC1 Modulates Transcription of a Core Cardiogenic Program in Human Cardiac Mesenchymal Stromal Cells Through a p53-Dependent Mechanism.

Histone deacetylase (HDAC) regulation is an essential process in myogenic differentiation. Inhibitors targeting the activity of specific HDAC family members have been shown to enhance the cardiogenic differentiation capacity of discrete progenitor cell types; a key property of donor cell populations contributing to their afforded benefits in cardiac cell therapy applications. The influence of HDAC inhibition on cardiac-derived mesenchymal stromal cell (CMC) transdifferentiation or the role of specific HDAC family members in dictating cardiovascular cell lineage specification has not been investigated. In the current study, the consequences of HDAC inhibition on patient-derived CMC proliferation, cardiogenic program activation, and cardiovascular differentiation/cell lineage specification were investigated using pharmacologic and genetic targeting approaches. Here, CMCs exposed to the pan-HDAC inhibitor sodium butyrate exhibited induction of a cardiogenic transcriptional program and heightened expression of myocyte and endothelial lineage-specific markers when coaxed to differentiate in vitro. Further, shRNA knockdown screens revealed CMCs depleted of HDAC1 to promote the induction of a cardiogenic transcriptional program characterized by enhanced expression of cardiomyogenic- and vasculogenic-specific markers, a finding which depended on and correlated with enhanced acetylation and stabilization of p53. Cardiogenic gene activation and elevated p53 expression levels observed in HDAC1-depleted CMCs were associated with improved aptitude to assume a cardiomyogenic/vasculogenic cell-like fate in vitro. These results suggest that HDAC1 depletion-induced p53 expression alters CMC cell fate decisions and identify HDAC1 as a potential exploitable target to facilitate CMC-mediated myocardial repair in ischemic cardiomyopathy. Stem Cells 2016;34:2916-2929.

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy.

RATIONALE: The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE: To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS: This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Type 2 Diabetes Dysregulates Glucose Metabolism in Cardiac Progenitor Cells.

Type 2 diabetes is associated with increased mortality and progression to heart failure. Recent studies suggest that diabetes also impairs reparative responses after cell therapy. In this study, we examined potential mechanisms by which diabetes affects cardiac progenitor cells (CPCs). CPCs isolated from the diabetic heart showed diminished proliferation, a propensity for cell death, and a pro-adipogenic phenotype. The diabetic CPCs were insulin-resistant, and they showed higher energetic reliance on glycolysis, which was associated with up-regulation of the pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). In WT CPCs, expression of a mutant form of PFKFB, which mimics PFKFB3 activity and increases glycolytic rate, was sufficient to phenocopy the mitochondrial and proliferative deficiencies found in diabetic cells. Consistent with activation of phosphofructokinase in diabetic cells, stable isotope carbon tracing in diabetic CPCs showed dysregulation of the pentose phosphate and glycero(phospho)lipid synthesis pathways. We describe diabetes-induced dysregulation of carbon partitioning using stable isotope metabolomics-based coupling quotients, which relate relative flux values between metabolic pathways. These findings suggest that diabetes causes an imbalance in glucose carbon allocation by uncoupling biosynthetic pathway activity, which could diminish the efficacy of CPCs for myocardial repair.