The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research.

OBJECTIVE: To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research. DESIGN: Prospective cohort study. SETTING: Not applicable. PATIENTS: A total of 50 in vitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July 2018. INTERVENTIONS: None. MAIN OUTCOME MEASURES: All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the ß-actin were used. Data were statistically analyzed by analysis of variance with the Scheffé multiple pairwise comparison for categorical variables and by linear regression for numerical variables. RESULTS: Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes. CONCLUSIONS: During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.

Variables associated with mitochondrial copy number in human blastocysts: what can we learn from trophectoderm biopsies?

OBJECTIVE: To study the potential variables that affect the mitochondrial DNA (mtDNA) content of trophectoderm (TE) cells in blastocysts that have undergone TE biopsy. DESIGN: Observational retrospective single-center analysis. SETTING: University-affiliated private in vitro fertilization center. PATIENT(S): A total of 465 consecutive preimplantation genetic screening (PGS) cycles of 402 women undergoing preimplantation genetic testing. INTERVENTION(S): Trophectoderm biopsy performed on blastocysts of women undergoing preimplantation genetic testing-aneuploidy (PGT-A). MAIN OUTCOME MEASURE(S): The mtDNA content in trophectoderm cells. RESULT(S): We checked the possible influence of patient characteristics, ovarian stimulation variables, embryo morphology, and embryo culture conditions on mtDNA values. Of all the analyzed variables, some such as body mass index (BMI), serum progesterone (P4), aneuploidy, and trophectoderm quality had an effect on mtDNA content in blastocysts. Body mass index had a small but positive effect on the mtDNA copy number; as the BMI values increased, the probability of women producing blastocysts with an mtDNA content above the median increased by 6%. For P4 serum concentration, an increase in P4 lowered the probability of blastocysts having values above the median by 39%. Embryo-associated variables such as TE quality and aneuploidy status appeared to affect the mtDNA copy number. For the aneuploid blastocysts, the probability of being above the median increased by 42%. Finally, blastocysts with poor quality TE had more chances of carrying higher mtDNA values. CONCLUSION(S): Summarizing, larger quantities of mtDNA in blastocysts are associated with the condition of aneuploidy and low quality TE, as well as being from women with high BMI values. Understanding the biological meaning of mtDNA content in human blastocysts and what factors may interfere with their values is fundamental. Other key gaps, such as whether a correlation exists between mtDNA content and mitochondrial mass and biogenesis in human TE cells, and whether this correlation can be extended to the inner cell mass, need to be further addressed. These questions are currently being investigated.

Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model.

BACKGROUND Cardioplegic arrest is a common procedure for many types of cardiac surgery, and different formulations have been proposed to enhance its cardio-protective effect. Hydrogen sulfide is an important signaling molecule that has cardio-protective properties. We therefore studied the cardio-protective effect of hydrogen sulfide in cardiac cell culture and its potential therapeutic use in combination with cardioplegia formulations. MATERIAL AND METHODS We added hydrogen sulfide donor GYY4137 to HL-1 cells to study its protective effect in nutrient starved conditions. In addition, we tested the potential use of GYY4137 when it is added into two different cardioplegia formulations: Cardi-Braun® solution and del Nido solution in an ex vivo Langendorff perfused rat hearts model. RESULTS We observed that eight-hour pre-treatment with GYY4137 significantly suppressed apoptosis in nutrient-starved HL-1 cells (28% less compared to untreated cells; p<0.05), maintained ATP content, and reduced protein synthesis. In ex vivo experiments, Cardi-Braun® and del Nido cardioplegia solutions supplemented with GYY4137 significantly reduced the pro-apoptotic protein caspase-3 content and preserved ATP content. Furthermore, GYY4137 supplemented cardioplegia solutions decreased the S-(5-adenosyl)-L-methionine/S-(adenosyl)-L-homocysteine ratio, reducing the oxidative stress in cardiac tissue. Finally, heart beating analysis revealed the preservation of the inter-beat interval and the heart rate in del Nido cardioplegia solution supplemented with GYY4137. CONCLUSIONS GYY4137 preconditioning preserved energetic state during starved conditions, attenuating the cardiomyocytes apoptosis in vitro. The addition of GYY4137 to cardioplegia solutions prevented apoptosis, ATP consumption, and oxidative stress in perfused rat hearts, restoring its electrophysiological status after cardiac arrest. These findings suggested that GYY4137 sulfide donor may improve the cardioplegia solution performance during cardiac surgery.

miRNA-1 and miRNA-133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of cardiac differentiation.

miRNA-1 (miR-1) and miRNA-133a (miR-133a) are muscle-specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR-1 and miR-133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR-1 and miR-133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)-mediated mesodermal and cardiac differentiation by gain- and loss-of-function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR-1 and miR-133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR-1 enhanced the cardiac differentiation of P19.CL6, while miR-133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR-1 and miR-133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR-1 and miR-133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.

Molecular disturbance underlies to arrhythmogenic cardiomyopathy induced by transgene content, age and exercise in a truncated PKP2 mouse model.

Arrhythmogenic cardiomyopathy (ACM) is a disorder characterized by a progressive ventricular myocardial replacement by fat and fibrosis, which lead to ventricular arrhythmias and sudden cardiac death. Mutations in the desmosomal gene Plakophilin-2 (PKP2) accounts for >40% of all known mutations, generally causing a truncated protein. In a PKP2-truncated mouse model, we hypothesize that content of transgene, endurance training and aging will be determinant in disease progression. In addition, we investigated the molecular defects associated with the phenotype in this model. We developed a transgenic mouse model containing a truncated PKP2 (PKP2-Ser329) and generated three transgenic lines expressing increasing transgene content. The pathophysiological features of ACM in this model were assessed. While we did not observe fibro-fatty replacement, ultrastructural defects were exhibited. Moreover, we observed transgene content-dependent development of structural (ventricle dilatation and dysfunction) and electrophysiological anomalies in mice (PR interval and QRS prolongation and arrhythmia induction). In concordance with pathological defects, we detected a content reduction and remodeling of the structural proteins Desmocollin-2, Plakoglobin, native Plakophilin-2, Desmin and ß-Catenin as well as the electrical coupling proteins Connexin 43 and cardiac sodium channel (Nav1.5). Surprisingly, we observed structural but not electrophysiological abnormalities only in trained and old mice. We demonstrated that truncated PKP2 provokes ACM in the absence of fibro-fatty replacement in the mouse. Transgene dose is essential to reveal the pathology, whereas aging and endurance training trigger limited phenotype. Molecular abnormalities underlay the structural and electrophysiological defects.

Aging and the environment affect gamete and embryo potential: can we intervene?

Optimal maturation of the oocyte depends on its environment and determines embryo competence, because the embryonic genome is not active until the cleavage stage and new mitochondria are not produced until blastulation. Adverse environmental factors include aging, andropause, oxidative stress, obesity, smoking, alcohol, and psychologic stress, whereas androgen supplementation, a prudent diet, exercise, nutritional supplements, and psychologic interventions have beneficial effects. Mitochondrial function and energy production deteriorate with age, adversely affecting ovarian reserve, chromosome segregation, and embryo competence. In aging mice, the mitochondrial cofactor coenzyme Q10 reverses most of these changes. Early human experience has been encouraging, although only a small study using a shorter duration of intervention compared with the murine model has been carried out. Mitochondrial metabolic stress can result in an abnormal compensatory increase in mitochondrial DNA, which can be assessed in biopsied blastomeres of trophectoderm as a predictive biomarker of implantation failure. Psychologic stress may reduce oocyte competence by shifting blood flow away from the ovary as part of the classic "fight or flight" physiologic response, and methods to reduce stress or the body's reaction to stress improve pregnancy success. Enhancing oocyte competence is a key intervention that promises to reduce the number of euploid embryos failing to produce viable deliveries.

Podocalyxin-like protein 1 is a relevant marker for human c-kit(pos) cardiac stem cells.

Cardiac progenitor cells (CPCs) from adult myocardium offer an alternative cell therapy approach for ischaemic heart disease. Improved clinical performance of CPCs in clinical trials requires a comprehensive definition of their biology and specific interactions with the environment. In this work we characterize specific human CPC surface markers and study some of their related functions. c-kit(pos) human CPCs (hCPCs) were characterized for cell surface marker expression, pluripotency, early and late cardiac differentiation markers and therapeutic activity in a rat model of acute myocardial infarction. The results indicate that hCPCs are a mesenchymal stem cell (MSC)-like population, with a similar immunoregulatory capacity. A partial hCPC membrane proteome was analysed by liquid chromatography-mass spectrometry/mass spectrometry and 36 proteins were identified. Several, including CD26, myoferlin and podocalyxin-like protein 1 (PODXL), have been previously described in other stem-cell systems. Suppression and overexpression analysis demonstrated that PODXL regulates hCPC activation, migration and differentiation; it also modulates their local immunoregulatory capacity. Therefore, hCPCs are a resident cardiac population that shares many features with hMSCs, including their capacity for local immunoregulation. Expression of PODXL appears to favour the immature state of hCPCs, while its downregulation facilitates their differentiation. Copyright © 2016 John Wiley & Sons, Ltd.

Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cells-mediated cardiac repair.

Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor-1 alpha (HIF-1α) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1α overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1α lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1α on MSC biology and suggests the stabilization of HIF-1α as a novel strategy for cellular therapies.

Cardiac stem cell genetic engineering using the alphaMHC promoter.

AIMS: Cardiac stem cells (CSCs) show potential as a cellular therapeutic approach to blunt tissue damage and facilitate reparative and regenerative processes after myocardial infarction. Despite multiple published reports of improvement, functional benefits remain modest using normal stem cells delivered by adoptive transfer into damaged myocardium. The goal of this study is to enhance survival and proliferation of CSCs that have undergone lineage commitment in early phases as evidenced by expression of proteins driven by the alpha-myosin heavy chain (alphaMHC) promoter. The early increased expression of survival kinases augments expansion of the cardiogenic CSC pool and subsequent daughter progeny. MATERIALS & METHODS: Normal CSCs engineered with fluorescent reporter protein constructs under control of the alphaMHC promoter show transgene protein expression, confirming activity of the promoter in CSCs. Cultured CSCs from both nontransgenic and cardiac-specific transgenic mice expressing survival kinases driven by the alphaMHC promoter were analyzed to characterize transgene expression following treatments to promote differentiation in culture. RESULTS & CONCLUSION: Therapeutic genes controlled by the alphaMHC promoter can be engineered into and expressed in CSCs and cardiomyocyte progeny with the goal of improving the efficacy of cardiac stem cell therapy.

Genetic evidence for a tumor suppressor role of HIF-2alpha.

The hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha are activated in hypoxic tumor regions. However, their role in tumorigenesis remains controversial, as tumor growth promoter and suppressor activities have been ascribed to HIF-1alpha, while the role of HIF-2alpha remains largely unknown. Here, we show that overexpression of HIF-2alpha in rat glioma tumors enhances angiogenesis but reduces growth of these tumors, in part by increasing tumor cell apoptosis. Moreover, siRNA knockdown of HIF-2alpha reduced apoptosis in hypoxic human malignant glioblastoma cells. Furthermore, inhibition of HIF by overexpression of a dominant-negative HIF transgene in glioma cells or HIF-2alpha deficiency in teratomas reduced vascularization but accelerated growth of these tumor types. These findings urge careful consideration of using HIF inhibitors as cancer therapeutic strategies.

Selective inactivation of p27(Kip1) in hematopoietic progenitor cells increases neointimal macrophage proliferation and accelerates atherosclerosis.

Excessive proliferation of immune cells and vascular smooth myocytes (VSMCs) contributes to atherosclerosis. We have previously shown that whole-body inactivation of the growth suppressor p27 exacerbates atherosclerosis in apolipoprotein E-null mice (apoE-/-), and this correlated with increased proliferation of arterial macrophages and VSMCs. In the present study, we postulated that targeted disruption of bone marrow (BM) p27 is sufficient to enhance arterial macrophage proliferation and atherosclerosis. To test this hypothesis, sublethally irradiated apoE-/- mice with an intact p27 gene received a BM transplant from either apoE-/- or p27-/-apoE-/- doubly deficient donor mice and challenged with a high-cholesterol diet. Compared with mice that received an apoE-/- BM transplant, reconstitution with p27-/-apoE-/- doubly deficient marrow increased the expression of proliferating cell nuclear antigen in neointimal macrophages and accelerated aortic atherosclerosis, and this correlated with augmented aortic expression of the inflammatory cytokines CCL2/MCP-1 (monocyte chemoattractant protein 1) and CCL5/RANTES (regulated on activation, normal T-cell expressed and secreted). Overall, these findings provide evidence that p27 deficiency in hematopoietic progenitor cells enhances the inflammatory/proliferative response induced by dietary cholesterol and accelerates atherosclerosis.

Coordinate control of proliferation and migration by the p27Kip1/cyclin-dependent kinase/retinoblastoma pathway in vascular smooth muscle cells and fibroblasts.

Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27Kip1-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27Kip1 inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27Kip1 mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders.

Role of the growth suppressor p27Kip1 during vascular remodeling.

At homeostasis, vascular cells display a very low proliferative rate and a scant migratory activity. However, hyperplastic growth and locomotion of vascular cells are a hallmark of vascular remodeling during several pathophysiological conditions (e.g., neovascularization, arteriosclerosis and restenosis post-angioplasty). Thus, a better understanding of the molecular mechanisms that control vascular cell proliferation and migration should facilitate the development of novel therapies to treat cardiovascular disease. In this review, we will discuss recent studies implicating the cell cycle regulatory protein p27Kip1 as a key modulator of vascular cell growth and locomotion in vitro and during vascular remodeling in vivo.

Distinct regulation of mitogen-activated protein kinases and p27Kip1 in smooth muscle cells from different vascular beds. A potential role in establishing regional phenotypic variance.

Excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in atherosclerotic plaque growth. In this study, we investigated whether SMCs from vessels with different atherogenicity exhibit distinct growth and migratory potential and investigated the underlying mechanisms. In fat-fed rabbits, we found increased cell proliferation and atheroma formation in the aortic arch versus the femoral artery. When examined in culture, SMCs isolated from the aortic arch (ASMCs) displayed a greater capacity for inducible proliferation and migration than paired cultures of femoral artery SMCs. Two lines of evidence suggested that distinct regulation of the growth suppressor p27(Kip1) (p27) contributes to establishing these phenotypic dissimilarities. First, p27 expression was comparably lower in ASMCs, which exhibited a higher fraction of p27 phosphorylated on Thr-187 and ubiquitinated. Second, forced p27 overexpression in ASMCs impaired their proliferative and migratory potential. We found that platelet-derived growth factor-BB-dependent induction of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway was comparably higher in ASMCs. Importantly, pharmacological inhibition of MAPKs increased p27 expression and attenuated ASMC proliferation and migration. In contrast, forced MAPK activation diminished p27 expression and markedly augmented femoral artery SMC proliferation and migration. We propose that intrinsic differences in the regulation of MAPKs and p27 play an important role in creating variance in the proliferative and migratory capacity of vascular SMCs, which might in turn contribute to establishing regional variability in atherogenicity.

Increased early atherogenesis in young versus old hypercholesterolemic rabbits by a mechanism independent of arterial cell proliferation.

We sought to determine the relative importance of aging and hypercholesterolemia on atherosclerosis. Although plasma cholesterol levels increased similarly in young and old rabbits fed an atherogenic diet for 2 months, aortic atherosclerotic lesions were more prominent in young animals. This finding was associated with an age-dependent reduction in the DNA-binding activity of the proinflammatory nuclear factor kappaB (NF-kappaB) in aortic tissue. Atherosclerotic lesions consisted mostly of macrophages, which displayed a similar proliferative response in both age groups. Independently of the age, medial cell proliferation was low and increased as a function of intimal lesion size. Thus, higher atherogenicity in young rabbits exposed to extreme hypercholesterolemia compared to old counterparts is associated with higher activity of NF-kappaB in the juvenile vessel wall without apparent age-dependent changes in arterial cell proliferation.