Effects of therapeutic ultrasound on the endothelial function of patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is characterized by endothelial dysfunction that causes micro- and macrovascular complications. Low intensity therapeutic ultrasound (LITUS) may improve endothelial function, but its effects have not been investigated in these patients. The aim of our study was to compare the effects of pulsed (PUT) and continuous (CUT) waveforms of LITUS on the endothelium-dependent vasodilation of T2DM patients. The present randomized crossover trial had a sample of twenty-three patients (7 men) diagnosed with T2DM, 55.6 (±9.1) years old, with a body mass index of 28.6 (±3.3) kg/m2. All patients were randomized and submitted to different waveforms (Placebo, CUT, and PUT) of LITUS and the arterial endothelial function was evaluated. The LITUS of 1 MHz was applied in pulsed (PUT: 20% duty cycle, 0.08 W/cm2 SATA), continuous (CUT: 0.4 W/cm2 SPTA), and Placebo (equipment off) types of waves during 5 min on the brachial artery. Endothelial function was evaluated using the flow-mediated dilation (FMD) technique. PUT (mean difference 2.08%, 95% confidence interval 0.65 to 3.51) and CUT (mean difference 2.32%, 95% confidence interval 0.89 to 3.74) increased the %FMD compared to Placebo. In the effect size analysis, PUT (d=0.65) and CUT (d=0.65) waveforms presented moderate effects in the %FMD compared to Placebo. The vasodilator effect was similar in the different types of waves. Pulsed and continuous waveforms of LITUS of 1 MHz improved the arterial endothelial function in T2DM patients.

Effects of therapeutic ultrasound on the endothelial function of patients with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is characterized by endothelial dysfunction that causes micro- and macrovascular complications. Low intensity therapeutic ultrasound (LITUS) may improve endothelial function, but its effects have not been investigated in these patients. The aim of our study was to compare the effects of pulsed (PUT) and continuous (CUT) waveforms of LITUS on the endothelium-dependent vasodilation of T2DM patients. The present randomized crossover trial had a sample of twenty-three patients (7 men) diagnosed with T2DM, 55.6 (±9.1) years old, with a body mass index of 28.6 (±3.3) kg/m2. All patients were randomized and submitted to different waveforms (Placebo, CUT, and PUT) of LITUS and the arterial endothelial function was evaluated. The LITUS of 1 MHz was applied in pulsed (PUT: 20% duty cycle, 0.08 W/cm2 SATA), continuous (CUT: 0.4 W/cm2 SPTA), and Placebo (equipment off) types of waves during 5 min on the brachial artery. Endothelial function was evaluated using the flow-mediated dilation (FMD) technique. PUT (mean difference 2.08%, 95% confidence interval 0.65 to 3.51) and CUT (mean difference 2.32%, 95% confidence interval 0.89 to 3.74) increased the %FMD compared to Placebo. In the effect size analysis, PUT (d=0.65) and CUT (d=0.65) waveforms presented moderate effects in the %FMD compared to Placebo. The vasodilator effect was similar in the different types of waves. Pulsed and continuous waveforms of LITUS of 1 MHz improved the arterial endothelial function in T2DM patients.

Effects of respiratory muscle training on parasympathetic activity in diabetes mellitus.

This study verified the effects of respiratory muscle training (RMT) on hemodynamics, heart rate (HR) variability, and muscle morphology in rats with streptozotocin-induced diabetes mellitus (DM). Thirty-six male Wistar rats were randomized into 4 groups and 34 completed the study: i) sham-sedentary (Sham-ST; n=9); ii) sham-RMT (Sham-RMT; n=9); iii) DM-sedentary (DM-ST; n=8); and iv) DM-RMT (DM-RMT; n=8). Hemodynamics were assessed by central cannulation, and R-R intervals were measured by electrocardiogram. In addition, the effects of RMT on the cross-sectional area of the diaphragm, anterior tibial, and soleus muscles were analyzed. The induction of DM by streptozotocin resulted in weight loss, hyperglycemia, reduced blood pressure, and attenuated left ventricular contraction and relaxation (P<0.05). We also observed a decrease in root mean square of successive differences between adjacent RR intervals (RMSSD) index and in the cross-sectional area of the muscles assessed, specifically the diaphragm, soleus, and anterior tibial muscles in diabetic rats (P<0.05). Interestingly, RMT led to an increase in RMSSD in rats with DM (P<0.05). The induction of DM produced profound deleterious changes in the diaphragmatic and peripheral muscles, as well as impairments in cardiovascular hemodynamics and autonomic control. Nevertheless, RMT may beneficially attenuate autonomic changes and improve parasympathetic modulation.

Effects of respiratory muscle training on parasympathetic activity in diabetes mellitus

This study verified the effects of respiratory muscle training (RMT) on hemodynamics, heart rate (HR) variability, and muscle morphology in rats with streptozotocin-induced diabetes mellitus (DM). Thirty-six male Wistar rats were randomized into 4 groups and 34 completed the study: i) sham-sedentary (Sham-ST; n=9); ii) sham-RMT (Sham-RMT; n=9); iii) DM-sedentary (DM-ST; n=8); and iv) DM-RMT (DM-RMT; n=8). Hemodynamics were assessed by central cannulation, and R-R intervals were measured by electrocardiogram. In addition, the effects of RMT on the cross-sectional area of the diaphragm, anterior tibial, and soleus muscles were analyzed. The induction of DM by streptozotocin resulted in weight loss, hyperglycemia, reduced blood pressure, and attenuated left ventricular contraction and relaxation (P<0.05). We also observed a decrease in root mean square of successive differences between adjacent RR intervals (RMSSD) index and in the cross-sectional area of the muscles assessed, specifically the diaphragm, soleus, and anterior tibial muscles in diabetic rats (P<0.05). Interestingly, RMT led to an increase in RMSSD in rats with DM (P<0.05). The induction of DM produced profound deleterious changes in the diaphragmatic and peripheral muscles, as well as impairments in cardiovascular hemodynamics and autonomic control. Nevertheless, RMT may beneficially attenuate autonomic changes and improve parasympathetic modulation.

Correction to: Flow-mediated dilation and heart failure: a review with implications to physical rehabilitation.

The scholarship support information in Acknowledgement was missing.

Flow-mediated dilation and heart failure: a review with implications to physical rehabilitation.

Endothelial dysfunction plays as an important role on mismatch responses that occur during exercise in patients with congestive heart failure (CHF). However, cardiac rehabilitation, a core component of management of CHF patients, can improve endothelial function, contributing to reduce the morbidity and mortality of these patients. The primary aims of this review were to describe the importance of flow-mediated dilatation (FMD) as a non-invasive validation tool to assess endothelial dysfunction and to highlight the relevance of scientific studies that evaluated the effects of exercise interventions on peripheral vascular endothelial function as measured by FMD in patients with CHF with both preserved and reduced ejection fraction.

Ultra-short-term heart rate variability during resistance exercise in the elderly.

Despite the appeal of ultra-short-term heart rate variability (HRV) methods of analysis applied in the clinical and research settings, the number of studies that have investigated HRV by analyzing R-R interval (RRi) recordings shorter than 5 min is still limited. Moreover, ultra-short-term HRV analysis has not been extensively validated during exercise and, currently, no indications exist for its applicability during resistance exercise. The aim of the present study was to compare ultra-short-term HRV analysis with standard short-term HRV analysis during low-intensity, dynamic, lower limb resistance exercise in healthy elderly subjects. Heart rate (HR) and RRi signals were collected from 9 healthy elderly men during discontinuous incremental resistance exercise consisting of 4-min intervals at low intensities (10, 20, 30, and 35% of 1-repetition maximum). The original RRi signals were segmented into 1-, 2-, and 3-min sections. HRV was analyzed in the time domain (root mean square of the of differences between adjacent RRi, divided by the number of RRi, minus one [RMSSD]), RRi mean value and standard deviation [SDNN] (percentage of differences between adjacent NN intervals that are greater than 50 ms [pNN50]), and by non-linear analysis (short-term RRi standard deviation [SD1] and long-term RRi standard deviation [SD2]). No significant difference was found at any exercise intensity between the results of ultra-short-term HRV analysis and the results of standard short-term HRV analysis. Furthermore, we observed excellent (0.70 to 0.89) to near-perfect (0.90 to 1.00) concordance between linear and non-linear parameters calculated over 1- and 2-min signal sections and parameters calculated over 3-min signal sections. Ultra-short-term HRV analysis appears to be a reliable surrogate of standard short-term HRV analysis during resistance exercise in healthy elderly subjects.

MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling.

Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Epigenetic memory in induced pluripotent stem cells.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Gene targeting in human pluripotent cells.

Mouse embryonic stem cells (mESCs) have the ability to differentiate into any cell type and can generate chimeric mice when transplanted into a host blastocyst. This remarkable potential, together with the development of robust gene targeting strategies in mESCs, were essential for establishing the mouse as the most widely used model organism in biomedical research. Recent advances have allowed the isolation of human embryonic stem cells and the derivation of induced pluripotent stem cells. Genetic tools similar to those proven routine in the mouse system are needed to realize the full potential of human pluripotent cells as disease models and putative therapeutics. Gene targeting in human cells, however, has proven to be more difficult, more time-consuming, and less robust than in mESCs. In this chapter, we discuss the strategies that have been used to allow specific genetic modifications in human pluripotent cells. We focus on the novel application of custom-engineered zinc-finger nucleases for gene targeting, which has promise to become a robust tool for efficient genetic manipulation of human pluripotent cells.

Stem cells, pluripotency and nuclear reprogramming.

Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by nuclear transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest as it has the potential to generate a source of patient-specific cells. This short review summarizes strategies to reprogram somatic cells to a pluripotent embryonic state and discuss the implications of this technology for transplantation medicine.

Reprogramming of somatic cell identity.

All mammalian somatic cells originate from a single fertilized cell, the zygote, and share identical genetic information despite the dramatic changes in cell structure and function that accompany organismal development. The genome is subjected to a wide array of epigenetic modifications during lineage specification, a process that contributes to the implementation and maintenance of specific gene expression programs in somatic cells. Nuclear transfer and cell-fusion experiments demonstrate that the epigenetic signature directing a cell identity can be erased and modified into that of another cell type. Furthermore, in the case of cloning, differentiated cells can be reprogrammed back to pluripotency to support the reexpression of all developmental programs. Recent breakthroughs highlight the importance of transcription factors as well as epigenetic modifiers in the establishment, maintenance, and rewiring of cell identity. By focusing on reprogramming of terminally differentiated lymphocytes, we review and highlight recent insights into the molecular mechanisms and cellular events potentially underlying programming and reprogramming of somatic cell identity in mammals.

Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice.

Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation.

Strategies to induce nuclear reprogramming.

The cloning of mammals from adult donor cells has demonstrated that the oocyte can reprogram a differentiated nucleus into a pluripotent embryonic state. Reprogramming of committed cells into pluripotent cells can also be achieved by the explantation of germ line cells and by the fusion of differentiated cells with embryonic cells. The future challenge will be to stably convert a differentiated cell into embryonic stem (ES) cells by the transient expression of defined genes. Recent findings suggest that the exposure of adult cells to a few defined factors can indeed induce a pluripotent-like state resembling that of ES cells. This approach may allow for the generation of patient-specific stem cells in order to study and treat degenerative diseases without recourse to nuclear transfer.

Dnmt1 expression in pre- and postimplantation embryogenesis and the maintenance of IAP silencing.

The methylation of intracisternal A-type particle (IAP) sequences is maintained during mouse embryogenesis. Methylation suppresses IAP expression and the potential for mutagenesis by retrotransposition, but it is not clear how methylation of these elements is maintained during the embryonic stages when the bulk of the genome is being demethylated. It has been suggested that the high levels of DNA methyltransferase-1 (Dnmt1) present during cleavage could be important for keeping IAPs methylated. To test this hypothesis, we combined mutant alleles of Dnmt1 with an agouti allele (A(iapy)), which provided a coat color readout for the methylation status of the IAP insertion in the agouti locus. We found that reduction in Dnmt1 levels directly impacted methylation at this locus, leading to stable transcriptional activation of the agouti gene in the adult. Specifically, the short maternal Dnmt1 protein was important in maintaining methylation at the A(iapy) locus in cleavage embryos, whereas the longer Dnmt1 isoform found in somatic cells was important in maintaining IAP methylation during the postimplantation stage. These results underscore the importance of maintaining proper maintenance of methylation patterns during gestation and suggest that interference with this process may stably affect gene expression patterns in the adult and may have profound phenotypic consequences.

Effects of cerebral ischemia in mice lacking DNA methyltransferase 1 in post-mitotic neurons.

DNA methylation is important for controlling gene expression and is catalyzed by DNA methyltransferase (Dnmt1) an enzyme abundant in brain. We recently demonstrated that mice expressing reduced levels of Dnmt1 are protected from cerebral ischemia. Here, we used the cre/loxP system to produce conditional mutants that lack Dnmt 1 in postmitotic neurons of the postnatal brain. We demonstrate that animals heterozygous for the conditional allele (Dnmt11lox/+) have significantly smaller infarcts following 1 h middle cerebral artery occlusion/reperfusion compared to their wildtype litters. Surprisingly, mice with a deletion of Dnmt1 in post-mitotic neurons (Dnmt11lox/c) were not protected. In conclusion, we demonstrate that reduced levels of Dnmt1, but not its absence, in post-mitotic neurons protect from ischemic brain injury.

Expression of Xist RNA is sufficient to initiate macrochromatin body formation.

MacroH2A1 is a histone variant that is found as a component of the inactive X chromosome where it is detected as a dense accumulation called a macrochromatin body (MCB). Macrochromatin bodies co-localize with Xist RNA, which is an untranslated RNA that is expressed exclusively from the inactive X chromosome of placental mammals. However, no studies to date have investigated whether Xist RNA expression is necessary or sufficient to cause the formation of MCBs. Here we show that expression of Xist RNA is sufficient to cause the formation of MCBs even when Xist is expressed from an inducible transgene at ectopic autosomal sites. Macrochromatin bodies form at sites of transgenic Xist expression in differentiating mouse ES cell lines and transgenic fibroblasts, but MCBs cannot form in undifferentiated ES cells even after prolonged Xist expression. The kinetics of MCB formation revealed that Xist expression precedes MCB formation and that differentiating ES cells undergo a rapid and synchronous transition that renders them competent to form MCBs. Once MCBs have formed, continued expression of Xist is required for their maintenance. These results show that Xist RNA and macroH2A1 function in a common pathway. Expression of Xist in a permissive nuclear environment is sufficient to initiate a chromatin-remodeling event culminating in the incorporation of macroH2A1. The results also strongly suggest the existence of additional regulatory factors for X inactivation that are regulated developmentally. In addition, we present evidence that macroH2A1 density is not simply a measure of the general degree of DNA compaction.