Left ventricular diastolic dysfunction in nonhuman primate model of dysmetabolism and diabetes.

BACKGROUND: Diabetes is one of the major risk factors for cardiomyopathy and heart failure with reduced ejection fraction (EF) and highly associated with left ventricular (LV) dysfunction in human. This study aimed 1) to noninvasively assess cardiac function using echocardiography; 2) to test the hypothesis that like diabetic human, cardiac function may also be compromised; in spontaneously developed obese, dysmetabolic and diabetic nonhuman primates (NHPs). METHODS: Cardiovascular functions were measured by noninvasive echocardiography in 28 control, 20 dysmetabolic/pre-diabetic and 41 diabetic cynomolgus monkeys based on fasting blood glucose and other metabolic status. RESULTS: The LV end-systolic volume (ESV) was higher while end-diastolic volume (EDV, 12 ± 5.7 mL) and EF (63 ± 12.8 %) significantly lower in the diabetic compared to control (14 ± 7 mL and 68 ± 9.8 %) group, respectively. The E/A ratio of LV trans-mitral peak flow rate during early (E) over late (A) diastole was significantly lower in the diabetic (1.19 ± 0.45) than control (1.44 ± 0.48) group. E-wave deceleration time (E DT) was prolonged in the diabetic (89 ± 41 ms) compared to control (78 ± 26 ms) group. Left atrial (LA) maximal dimension (LADmax) was significantly greater in the diabetic (1.3 ± 0.17 cm) than control (1.1 ± 0.16 cm) group. Biochemical tests showed that total cholesterol and LDL were significant higher in the diabetic (167 ± 63 and 69 ± 37 mg/dL) than both pre-diabetic (113 ± 37 and 41 ± 23 mg/dL) and control (120 ± 28 and 41 ± 17 mg/dL) groups, respectively. Multivariable logistic regression analysis demonstrated that LV systolic (reduced EF) and diastolic (abnormal E/A ratio) dysfunctions are significantly correlated with aging and hyperglycemia. Histopathology examination of the necropsy heart revealed inflammatory infiltration, cardiomyocyte hypertrophy and fragmentation, indicating the myocardial ischemia and remodeling which is consistent with the LV dysfunction phenotype. CONCLUSIONS: Using noninvasive echocardiography, the present study demonstrated for the first time that dysmetabolic and diabetic NHPs are associated with LV systolic (increased ESV, decreased EF, etc.) and diastolic (decreased EDV and E/A ratio, prolonged E DT, etc.) dysfunctions, accompanied by LA hypertrophic remodeling (increased LADmax), the phenotypes similarly to those found in diabetic patients. Thus, spontaneously developed dysmetabolic and diabetic NHPs is a highly translatable model to human diseases not only in the pathogenic mechanisms but also can be used for testing novel therapies for cardiometabolic disorders.

Germline and somatic imprinting in the nonhuman primate highlights species differences in oocyte methylation.

Genomic imprinting is an epigenetic mechanism resulting in parental allele-specific gene expression. Defects in normal imprinting are found in cancer, assisted reproductive technologies, and several human syndromes. In mouse models, germline-derived DNA methylation is shown to regulate imprinting. Though imprinting is largely conserved between mammals, species- and tissue-specific domains of imprinted expression exist. Using the cynomolgus macaque (Macaca fascicularis) to assess primate-specific imprinting, we present a comprehensive view of tissue-specific imprinted expression and DNA methylation at established imprinted gene clusters. For example, like mouse and unlike human, macaque IGF2R is consistently imprinted, and the PLAGL1, INPP5F transcript variant 2, and PEG3 imprinting control regions are not methylated in the macaque germline but acquire this post-fertilization. Methylome data from human early embryos appear to support this finding. These suggest fundamental differences in imprinting control mechanisms between primate species and rodents at some imprinted domains, with implications for our understanding of the epigenetic programming process in humans and its influence on disease.

Alterations to DNA methylation and expression of CXCL14 are associated with suboptimal birth outcomes.

CXCL14 is a chemokine that has previously been implicated in insulin resistance in mice. In humans, the role of CXCL14 in metabolic processes is not well established, and we sought to determine whether CXCL14 is a risk susceptibility gene important in fetal programming of metabolic disease. For this purpose, we investigated whether CXCL14 is differentially regulated in human umbilical cords of infants with varying birth weights. We found an elevated expression of CXCL14 in human low birth weight (LBW) cords, as well as in cords from nutritionally restricted Macaca fascicularis macaques. To further analyze the regulatory mechanisms underlying the expression of CXCL14, we examined CXCL14 in umbilical cord-derived mesenchymal stem cells (MSCs) that provide an in vitro cell-based system amenable to experimental manipulation. Using both whole frozen cords and MSCs, we determined that site-specific CpG methylation in the CXCL14 promoter is associated with altered expression, and that changes in methylation are evident in LBW infant-derived umbilical cords that may indicate future metabolic compromise through CXCL14.

Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions.

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of "simple" cancer-associated chromosome deletions.

Improved granulocyte colony-stimulating factor mobilization of hemopoietic progenitors using cytokine combinations in primates.

Peripheral blood stem cells (PBSCs), usually mobilized with granulocyte colony-stimulating factor (G-CSF) alone or in combination with chemotherapy, are the preferred source of cells for hemopoietic stem cell transplantation. Up to 25% of otherwise eligible transplant recipients fail to harvest adequate PBSCs. Therefore it is important to investigate existing and novel reagents to improve PBSC mobilization. Because of marked interindividual variation in humans, we developed a robust nonhuman primate model that allows the direct comparison of the efficacy of two PBSC mobilization regimens within the same animal. Using this model, we compared pegylated G-CSF (pegG-CSF) with standard G-CSF and compared the combination of G-CSF and pegylated megakaryocyte growth and development factor (pegMGDF) with G-CSF plus stem cell factor (SCF) by measuring the levels of CD34(+) cells, colony-forming cells (CFCs), and SCID repopulating cells (SRCs) before and after cytokine administration. Mobilization of CD34(+) cells, CFCs and SRCs using pegG-CSF achieved similar levels to those resulting from 5 days of standard G-CSF. The combination of G-CSF+pegMGDF mobilized progenitors to levels similar to G-CSF+SCF but greater than standard G-CSF for CD34(+) cells and CFC. This first direct comparison of PBSC mobilization in individual primates demonstrates that peg-G-CSF is equivalent to daily G-CSF and that the addition of pegMGDF to G-CSF improves mobilization. In light of the development of new thrombopoietin agonists, these data offer the potential for improved stem cell mobilization strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) show great promise for ex vivo gene and cell-mediated therapies. The immunophenotype and in vitro differentiation capacity of primary baboon MSCs was demonstrated to be near-identical to that observed in human MSCs. To optimize gene transfer efficiency, we compared the efficiency of serotypes 1, 2, 3, 4, 5, 6, and 8 of adeno-associated virus (AAV) vectors for their ability to mediate transduction of human and baboon MSCs. AAV serotype 2 vectors were the most efficient in transducing MSCs from humans and baboons. As a reference, human Ad293 cells were transduced with these seven AAV serotypes, and were found to have the highest transduction levels followed by baboon MSCs, and then human MSCs. The order of increasing transduction efficiency for the serotypes tested was similar for human and baboon MSCs, but was different for human Ad293 cells. The transduction efficiency of MSCs isolated from different individuals was comparable within the same species. We also demonstrated that baboon MSCs transduced with AAV serotype 2 vectors retain their potential to differentiate into adipocytes in vitro, and can incorporate into injured muscle tissue of NODSCID mice in vivo. We detected beta-galactosidase reporter gene expression in host muscle tissue for up to 9 weeks in this study, indicating engraftment of transduced baboon MSCs and sustained transgene expression in vivo.