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.

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

AIMS: Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. METHODS AND RESULTS: In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. CONCLUSION: These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease.

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.

Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells.

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship that is essential for maintaining normal development and function in the heart. Little is known about the mechanisms that regulate cardiac and endothelial crosstalk, particularly in situations of acute stress when local active processes are required to regulate endothelial function. We examined whether CM-derived exosomes could modulate endothelial function. Under conditions of glucose deprivation, immortalized H9C2 cardiomyocytes increase their secretion of exosomes. CM-derived exosomes are loaded with a broad repertoire of miRNA and proteins in a glucose availability-dependent manner. Gene Ontology (GO) analysis of exosome cargo molecules identified an enrichment of biological process that could alter EC activity. We observed that addition of CM-derived exosomes to ECs induced changes in transcriptional activity of pro-angiogenic genes. Finally, we demonstrated that incubation of H9C2-derived exosomes with ECs induced proliferation and angiogenesis in the latter. Thus, exosome-mediated communication between CM and EC establishes a functional relationship that could have potential implications for the induction of local neovascularization during acute situations such as cardiac injury.

Mitochondrial DNA content as a viability score in human euploid embryos: less is better.

OBJECTIVE: To investigate the clinical relevance of mitochondrial DNA (mtDNA) content as a viability score in human euploid embryos. DESIGN: Retrospective analysis of mtDNA content of transferred euploid embryos. SETTING: Reproductive genetics laboratory. PATIENT(S): Single-embryo transfer in 270 patients who underwent preimplantation genetic screening (205 day-3 blastomere biopsies, and 65 day-5 trophectoderm biopsies), and 10 patients with double-embryo transfer (male-female). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Normalized mtDNA content versus nuclear DNA (nDNA) from transferred euploid embryos. RESULT(S): A high mtDNA copy number in euploid embryos is indicative of lower embryo viability and implantation. Using the normalized mtDNA content, we created the mitochondrial score or Mitoscore (Ms). Day-3 embryos with <34 (MsA) had an implantation rate (IR) of 59% (n = 51); those with 34-52 (MsB) had an IR of 44% (n = 52); those with 52-97 (MsC) had an IR of 42% (n = 50); and those with >97 (MsD) had an IR of 25% (n = 52). Embryos with Ms >160 (n = 22) never implanted. Day-5 embryos with <18.19 (MsA) had an IR of 81%; those with 18.19-24.15 (MsB) had an IR of 50% (n = 16); those with 24.15-50.58 (MsC) had an IR of 62% (n = 16); and those with levels >50.58 (MsD) had an IR of 18% (n = 17). Embryos with levels >60 (n = 7) never implanted. CONCLUSION(S): An increased amount of mtDNA in euploid embryos is related to poor implantation potential and may be indicative of reduced metabolic fuel during oocyte maturation. We are implementing Ms in our preimplantation genetic screening platform to prospectively analyze its clinical relevance.

Converting a Problem into an Opportunity: mtDNA Heteroplasmy Shift.

The transmission of mitochondrial DNA (mtDNA) disease from a mother with a heteroplasmic mtDNA mutation to her children is unpredictable. In a recent issue of Cell, Reddy et al. (2015) present the potential for mitochondrial-targeted nucleases to remove mutated mtDNA through the induction of heteroplasmy shift in oocytes and zygotes.

miR-133a enhances the protective capacity of cardiac progenitors cells after myocardial infarction.

miR-133a and miR-1 are known as muscle-specific microRNAs that are involved in cardiac development and pathophysiology. We have shown that both miR-1 and miR-133a are early and progressively upregulated during in vitro cardiac differentiation of adult cardiac progenitor cells (CPCs), but only miR-133a expression was enhanced under in vitro oxidative stress. miR-1 was demonstrated to favor differentiation of CPCs, whereas miR-133a overexpression protected CPCs against cell death, targeting, among others, the proapoptotic genes Bim and Bmf. miR-133a-CPCs clearly improved cardiac function in a rat myocardial infarction model by reducing fibrosis and hypertrophy and increasing vascularization and cardiomyocyte proliferation. The beneficial effects of miR-133a-CPCs seem to correlate with the upregulated expression of several relevant paracrine factors and the plausible cooperative secretion of miR-133a via exosomal transport. Finally, an in vitro heart muscle model confirmed the antiapoptotic effects of miR-133a-CPCs, favoring the structuration and contractile functionality of the artificial tissue.

A transgenic Xenopus laevis reporter model to study lymphangiogenesis.

The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

The decay of stem cell nourishment at the niche.

One of the main features of human aging is the loss of adult stem cell homeostasis. Organs that are very dependent on adult stem cells show increased susceptibility to aging, particularly organs that present a vascular stem cell niche. Reduced regenerative capacity in tissues correlates with reduced stem cell function, which parallels a loss of microvascular density (rarefraction) and plasticity. Moreover, the age-related loss of microvascular plasticity and rarefaction has significance beyond metabolic support for tissues because stem cell niches are regulated co-ordinately with the vascular cells. In addition, microvascular rarefaction is related to increased inflammatory signals that may negatively regulate the stem cell population. Thus, the processes of microvascular rarefaction, adult stem cell dysfunction, and inflammation underlie the cycle of physiological decline that we call aging. Observations from new mouse models and humans are discussed here to support the vascular aging theory. We develop a novel theory to explain the complexity of aging in mammals and perhaps in other organisms. The connection between vascular endothelial tissue and organismal aging provides a potential evolutionary conserved mechanism that is an ideal target for the development of therapies to prevent or delay age-related processes in humans.

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.

Complement anaphylatoxins C3a and C5a induce a failing regenerative program in cardiac resident cells. Evidence of a role for cardiac resident stem cells other than cardiomyocyte renewal.

Cardiac healing, which follows myocardial infarction, is a complex process guided by intricate interactions among different components. Some resident cell populations with a potential role in cardiac healing have already been described in cardiac tissues. These non-cardiomyocyte cell subsets, globally described as cardiac pluripotent/progenitor cells (CPCs), are able to differentiate into all three major cardiac cell lineages (endothelial, smooth muscle and cardiomyocyte cells) in experimental settings. Nevertheless, physiological cardiac healing results in a fibrous scar, which remains to be fully modelled experimentally. Since a role for complement anaphylatoxins (C3a and C5a) has been described in several regeneration/repair processes, we examined the effects that C3a and C5a exert on a defined population of CPCs. We found that C3a and C5a are able to enhance CPC migration and proliferation. In vitro studies showed that this effect is linked to activation of telomerase mRNA and partial preservation of telomere length, in an NFκB-dependent manner. In addition, anaphylatoxin signalling modulates the CPC phenotype, increasing myofibroblast differentiation and reducing endothelial and cardiac gene expression. These findings may denote that C3a and C5a are able to maintain/increase the cardiac stem cell pool within the heart, whilst simultaneously facilitating and modulating resident cell differentiation. We found that this modulation was directed towards scar forming cells, which increased fibroblast/myofibroblast generation and suggests that both these anaphylatoxins could play a relevant role in the damage-coupled activation of resident cells, and regulation of the cardiac healing process after injury.

Deficient p27 phosphorylation at serine 10 increases macrophage foam cell formation and aggravates atherosclerosis through a proliferation-independent mechanism.

OBJECTIVE: Genetic ablation of the growth suppressor p27(Kip1) (p27) in the mouse aggravates atherosclerosis coinciding with enhanced arterial cell proliferation. However, it is unknown whether molecular mechanisms that limit p27's protective function contribute to atherosclerosis development and whether p27 exerts proliferation-independent activities in the arterial wall. This study aims to provide insight into both questions by investigating the role in atherosclerosis of p27 phosphorylation at serine 10 (p27-phospho-Ser10), a major posttranslational modification of this protein. METHODS AND RESULTS: Immunoblotting studies revealed a marked reduction in p27-phospho-Ser10 in atherosclerotic arteries from apolipoprotein E-null mice, and expression of the nonphosphorylatable mutant p27Ser10Ala, either global or restricted to bone marrow, accelerated atherosclerosis. p27Ser10Ala expression did not affect cell proliferation in early and advanced atheroma but activated RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling and promoted macrophage foam cell formation in a ROCK-dependent manner. Supporting the clinical relevance of these findings, human atherosclerotic coronary arteries exhibited a prominent reduction in p27-phospho-Ser10 and increased ezrin/radixin/moesin protein phosphorylation, a marker of RhoA/ROCK activation. CONCLUSION: Scarce phosphorylation of p27 at Ser10 is a hallmark of human and mouse atherosclerosis and promotes disease progression in mice. This proatherogenic effect is mediated by a proliferation-independent mechanism that involves augmented foam cell formation owing to increased RhoA/ROCK activity. These findings unveil a new atheroprotective action of p27 and identify p27-phospho-Ser10 as an attractive target for the treatment of atherosclerosis.

The extracellular matrix metalloproteinase inducer EMMPRIN is a target of nitric oxide in myocardial ischemia/reperfusion.

Nitric oxide (NO) is an important defense against myocardial ischemia/reperfusion (I/R) injury. Although matrix metalloproteinase (MMP)-mediated necrosis of cardiac myocytes is well characterized, the role of inducible NO synthase (iNOS)-derived NO in this process is poorly understood. I/R injury was increased in iNOS-deficient mice and in mice treated with 1400 W (a pharmacological iNOS inhibitor) and was associated with significantly increased expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and EMMPRIN-associated MMPs. Transcriptional activity of an EMMPRIN luciferase promoter reporter expressed in cardiac myocytes was inhibited by NO in a cGMP-dependent manner, and this transcriptional inhibition was abolished by mutation of a putative E2F site. Consistent with these findings, EMMPRIN null mice, in which iNOS is normally induced, are partially protected against I/R injury. Pharmacological inhibition of iNOS in EMMPRIN null mice had no additional protective effect, suggesting that EMMPRIN is a downstream target of NO. Administration of anti-EMMPRIN neutralizing antibodies partly reduced the excess heart damage and MMP-9 expression induced by I/R in iNOS null mice, indicating that regulation of EMMPRIN is an important mechanism of NO-mediated cardioprotection.