Karen Hensen

Recovery of Regional but Not Global Contractile Function by the Direct Intramyocardial Autologous Bone Marrow Transplantation Results From a Randomized Controlled Clinical Trial

Background— Recent trials have shown that intracoronary infusion of bone marrow cells (BMCs) improves functional recovery after acute myocardial infarction. However, whether this treatment is effective in heart failure as a consequence of remodeling after organized infarcts remains unclear. In this randomized trial, we assessed the hypothesis that direct intramyocardial injection of autologous mononuclear bone marrow cells during coronary artery bypass graft (CABG) could improve global and regional left ventricular ejection fraction (LVEF) at 4-month follow-up. Methods and Results— Twenty patients (age 64.8±8.7; 17 male, 3 female) with a postinfarction nonviable scar, as assessed by thallium (Tl) scintigraphy and cardiac magnetic resonance imaging (MRI), scheduled for elective CABG, were included. They were randomized to a control group (n =10, CABG only) or a BMC group (CABG and injection of 60.106±31.106 BMC). Primary end points were global LVEF change and wall thickening changes in the infarct area from baseline to 4-month follow-up, as measured by MRI. Changes in metabolic activity were measured by Tl scintigraphy and expressed as a score with a range from 0 to 4, corresponding to percent of maximal myocardial Tl uptake (4 indicates <50%, nonviable scar; 3, 50% to 60%; 2, 60% to 70%; 1, 70% to 80%; 0>80%). Global LVEF at baseline was 39.5±5.5% in controls and 42.9±10.3% in the BMC group (P=0.38). At 4 months, LVEF had increased to 43.1±10.9% in the control group and to 48.9±9.5% in the BMC group (P=0.23). Systolic thickening had improved from −0.6±1.3 mm at baseline to 1.8±2.6 mm at 4 months in the cell-implanted scars, whereas nontreated scars remained largely akinetic (−0.5±2.0 mm at baseline compared with 0.4±1.7 mm at 4 months, P=0.007 control versus BMC-treated group at 4 months). Defect score decreased from 4 to 3.3±0.9 in the BMC group and to 3.7±0.4 in the control group (P=0.18). Conclusions— At 4 months, there was no significant difference in global LVEF between both groups, but a recovery of regional contractile function in previously nonviable scar was observed in the BMC group.

Transcriptional Activation Capacity of the Novel PLAG Family of Zinc Finger Proteins

We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12. PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA. To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast. Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region. This effect is less profound in epithelial cells. These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes.

Microarray screening for target genes of the proto-oncogene PLAG1

PLAG1 is a proto-oncogene whose ectopic expression can trigger the development of pleomorphic adenomas of the salivary glands and of lipoblastomas. As PLAG1 is a transcription factor, able to activate transcription through the binding to the consensus sequence GRGGC(N)6–8GGG, its ectopic expression presumably results in the deregulation of target genes, leading to uncontrolled cell proliferation. The identification of PLAG1 target genes is therefore a crucial step in understanding the molecular mechanisms involved in PLAG1-induced tumorigenesis. To this end, we analysed the changes in gene expression caused by the conditional induction of PLAG1 expression in fetal kidney 293 cell lines. Using oligonucleotide microarray analyses of about 12u2009000 genes, we consistently identified 47 genes induced and 12 genes repressed by PLAG1. One of the largest classes identified as upregulated PLAG1 targets consists of growth factors such as the insulin-like growth factor II and the cytokine-like factor 1. The in silico search for PLAG1 consensus sequences in the promoter of the upregulated genes reveals that a large proportion of them harbor several copies of the PLAG1-binding motif, suggesting that they represent direct PLAG1 targets. Our approach was complemented by the comparison of the expression profiles of pleomorphic adenomas induced by PLAG1 versus normal salivary glands. Concordance between these two sets of experiments pinpointed 12 genes that were significantly and consistently upregulated in pleomorphic adenomas and in PLAG1-expressing cells, identifying them as putative PLAG1 targets in these tumors.

Human bone marrow stem cells co-cultured with neonatal rat cardiomyocytes display limited cardiomyogenic plasticity.

BACKGROUND AIMSnThis study investigated whether neonatal rat cardiomyocytes (NRCM), when co-cultured, can induce transdifferentiation of either human mesenchymal stromal cells (MSC) or hematopoietic stem cells (HSC) into cardiomyocytes. Stem cells were obtained from patients with ischemic heart disease.nnnMETHODSnEx vivo-expanded MSC or freshly isolated HSC were used to set-up a co-culture system between NRCM and MSC or HSC. 5-azacytidin (5-aza) or dimethylsulfoxide (DMSO) was used as differentiation-inducing factor. Co-cultured stem cells were separated from NRCM by flow sorting, and cardiac gene expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular morphology was analyzed by immunofluorescence and transmission electron microscopy (TEM).nnnRESULTSnCo-culturing MSC induced expression of troponin T and GATA-4. However, no expression of alpha-actinin, myosin heavy chain or troponin I was detected. In the case of HSC, only expression of troponin T could be induced. Immunofluorescence and TEM confirmed the absence of sarcomeric organization in co-cultured MSC and HSC. Adding 5-aza or DMSO to the co-cultures did not influence differentiation.nnnCONCLUSIONSnThis in vitro co-culture study obtained no convincing evidence of transdifferentiation of either MSC or HSC into functional cardiomyocytes. Nevertheless, induction of troponin T was observed in MSC and HSC, and GATA-4 in MSC. However, no morphologic changes could be detected by immunofluorescence or by TEM. These data could explain why only limited functional improvement was reported in clinical stem cell trials.

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study

In the past, clinical trials transplanting bone marrow–derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit+ CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit+ CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.

Targeted disruption of the murine Plag1 proto‐oncogene causes growth retardation and reduced fertility

The pleomorphic adenoma gene 1 (Plag1) proto‐oncogene encodes a transcription factor and is implicated in human tumorigenesis via ectopic overexpression. No information is available about its developmental role. To address this, a Plag1–/– mouse strain was generated and it appears that Plag1‐deficient mice are viable. No anatomical differences are obvious at birth, except that the weight of Plag1–/– mice is significantly lower in comparison to control litter mates. This early growth retardation is maintained throughout adult life with proportionally smaller organs except for the disproportionally small seminal vesicles and ventral prostate; however, plasma testosterone levels in males were not affected. Furthermore, fertility of both male and female Plag1–/– is reduced. Northern blot analysis revealed that Plag1 is developmentally regulated with high overall fetal expression levels, which drop after birth. Furthermore, Plag1 is differentially expressed and is readily detectable in the reproductive organs and pituitary. Expression of growth regulatory Igf2, a known target gene of Plag1 in tumorigenesis, was not affected in Plag1–/– embryos and pups. The general morphology and histology of the size‐reduced pituitaries was not affected. Our results establish that Plag1 disruption in mouse differentially affects pre‐ and postnatal growth and development of organs, with reproductive repercussions.

Clinical benefits of the addition of lower extremity low-intensity resistance muscle training to early aerobic endurance training intervention in patients with coronary artery disease: a randomized controlled trial.

OBJECTIVEnMuscle resistance training is often combined with aerobic endurance training during rehabilitation of patients with coronary artery disease. However, the clinical effects of additional lower-extremity low-intensity muscle resistance training during early rehabilitation (within the first month after coronary revascularization) in patients with coronary artery disease remain unclear.nnnDESIGNnProspective randomized controlled trial.nnnSUBJECTSnSixty patients with coronary artery disease.nnnMETHODSnSubjects were randomly assigned to early aerobic endurance training (nu2009=u200930) or combined aerobic endurance and resistance muscle training (nu2009=u200930). Subjects performed 18 (standard deviation 2) exercise sessions (at 65% VO(2peak), for 40 mins/session). In resistance muscle training, additional low-intensity (12-20 repetition maximum) resistance muscle exercises were performed. The following parameters were evaluated: exercise capacity, body composition, blood lipid profile, glycaemic control, blood endothelial progenitor cell and cytokine content, and muscle performance.nnnRESULTSnA total of 47 patients with coronary artery disease completed the intervention. Total body lean tissue mass tended to increase with greater magnitude (pu2009=u20090.07), and blood high-density lipid cholesterol content increased with significantly greater magnitude in resistance muscle training (pu2009<u20090.05), compared with aerobic endurance training. Maximal exercise capacity, ventilatory threshold, and muscle performance increased, and steady-state exercise respiratory exchange ratio, and adipose tissue mass reduced significantly (p <u20090.05), without differences between groups (p <u20090.05).nnnCONCLUSIONnIn early aerobic endurance training intervention in patients with coronary artery disease, additional low-intensity resistance muscle training contributes to a greater increase in blood high-density lipid cholesterol content, and tends to affect lean tissue mass.

From Bone Marrow to Cardiac Atrial Appendage Stem Cells for Cardiac Repair: A Review

Traditionally the heart is considered a terminally differentiated organ. However, at the beginning of this century increased mitotic activity was reported in ischemic and idiopathic dilated cardiomyopathy hearts, compared to healthy controls, underscoring the potential of regeneration after injury. Due to the presence of adult stem cells in bone marrow and their purported ability to differentiate into other cell lineages, this cell population was soon estimated to be the most suited candidate for cardiac regeneration. Clinical trials with autologous bone marrow-derived mononuclear cells, using either an intracoronary or direct intramyocardial injection approach consistently showed only minor improvement in global left ventricular ejection fraction. This was explained by their limited cardiomyogenic differentiation potential. To obtain more convincing improvement in cardiac function, based on true myocardial regeneration, the focus of research has shifted towards resident cardiac progenitor cells. Several isolation procedures have been described: the c-kit surface marker was the first to be used, however experimental research has clearly shown that c-kit+ cells only marginally contribute to regeneration post myocardial infarction. Sphere formation was used to isolate the so-called cardiosphere derived cells (CDC), and also in this cell population cardiomyogenic differentiation is a rare event. Recently a new type of stem cells derived from atrial tissue (cardiac atrial stem cells - CASCs) was identified, based on the presence of the enzyme aldehyde dehydrogenase (ALDH). Those cells significantly improve both regional and global LV ejection fraction, based on substantial engraftment and consistent differentiation into mature cardiomyocytes (98%).