Laurence H. Kedes

Systemic Delivery of Bone Marrow–Derived Mesenchymal Stem Cells to the Infarcted Myocardium Feasibility, Cell Migration, and Body Distribution

Background—Systemic delivery of bone marrow–derived mesenchymal stem cells (BM-MSCs) is an attractive approach for myocardial repair. We aimed to test this strategy in a rat model after myocardial infarction (MI). Methods and Results—BM-MSCs were obtained from rat bone marrow, expanded in vitro to a purity of >50%, and labeled with 99mTc exametazime, fluorescent dye, LacZ marker gene, or bromodeoxyuridine. Rats were subjected to MI by transient coronary artery occlusion or to sham MI. 99mTc-labeled cells (4×106) were transfused into the left ventricular cavity of MI rats either at 2 or 10 to 14 days after MI and were compared with sham-MI rats or MI rats treated with intravenous infusion. Gamma camera imaging and isolated organ counting 4 hours after intravenous infusion revealed uptake of the 99mTc-labeled cells mainly in the lungs, with significantly smaller amounts in the liver, heart, and spleen. Delivery by left ventricular cavity infusion resulted in drastically lower lung uptake, better uptake in the heart, and specifically higher uptake in infarcted compared with sham-MI hearts. Histological examination at 1 week after infusion identified labeled cells either in the infarcted or border zone but not in remote viable myocardium or sham-MI hearts. Labeled cells were also identified in the lung, liver, spleen, and bone marrow. Conclusions—Systemic intravenous delivery of BM-MSCs to rats after MI, although feasible, is limited by entrapment of the donor cells in the lungs. Direct left ventricular cavity infusion enhances migration and colonization of the cells preferentially to the ischemic myocardium.

Molecular Mechanisms of Myogenic Coactivation by p300: Direct Interaction with the Activation Domain of MyoD and with the MADS Box of MEF2C

By searching for molecules that assist MyoD in converting fibroblasts to muscle cells, we have found that p300 and CBP, two related molecules that act as transcriptional adapters, coactivate the myogenic basic-helix-loop-helix (bHLH) proteins. Coactivation by p300 involves novel physical interactions between p300 and the amino-terminal activation domain of MyoD. In particular, disruption of the FYD domain, a group of three amino acids conserved in the activation domains of other myogenic bHLH proteins, drastically diminishes the transactivation potential of MyoD and abolishes both p300-mediated coactivation and the physical interaction between MyoD and p300. Two domains of p300, at its amino and carboxy terminals, independently function to both mediate coactivation and physically interact with MyoD. A truncated segment of p300, unable to bind MyoD, acts as a dominant negative mutation and abrogates both myogenic conversion and transactivation by MyoD, suggesting that endogenous p300 is a required coactivator for MyoD function. The p300 dominant negative peptide forms multimers with intact p300. p300 and CBP serve as coactivators of another class of transcriptional activators critical for myogenesis, myocyte enhancer factor 2 (MEF2). In fact, transactivation mediated by the MEF2C protein is potentiated by the two coactivators, and this phenomenon is associated with the ability of p300 to interact with the MADS domain of MEF2C. Our results suggest that p300 and CBP may positively influence myogenesis by reinforcing the transcriptional autoregulatory loop established between the myogenic bHLH and the MEF2 factors.

Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat—angiogenesis and angioma formation☆

OBJECTIVES The effects of direct intramyocardial injection of the plasmid encoding vascular endothelial growth factor (phVEGF165) in the border zone of myocardial infarct tissue in rat hearts were investigated. BACKGROUND Controversy exists concerning the ability of VEGF to induce angiogenesis and enhance coronary flow in the myocardium. METHODS Sprague-Dawley rats received a ligation of the left coronary artery to induce myocardial infarction (MI). At 33.1 +/- 6.5 days, the rats were injected with phVEGF165 at one location and control plasmid at a second location (500 microg DNA, n = 24) or saline (n = 16). After 33.1 +/- 5.7 days, the hearts were excised for macroscopic and histologic analysis. Regional blood flow ratios were measured in 18 rats by radioactive microspheres. RESULTS phVEGF165-treated sites showed macroscopic angioma-like structures at the injection site while control DNA and saline injection sites did not. By histology, 21/24 phVEGF165-treated hearts showed increased focal epicardial blood vessel density and angioma-like formation. Quantitative morphometric evaluation in 20 phVEGF165-treated hearts revealed 44.4 +/- 10.5 vascular structures per field in phVEGF165-treated hearts versus 21.4 +/- 4.7 in control DNA injection sites (p < 0.05). Regional myocardial blood flow ratios between the injection site and noninfarcted area did not demonstrate any difference between phVEGF,165-treated hearts (0.9 +/- 0.2) and saline-treated hearts (0.7 +/- 0.1). CONCLUSIONS Injection of DNA for VEGF in the border zone of MI in rat hearts induced angiogenesis. Angioma formation at the injection sites did not appear to contribute to regional myocardial blood flow, which may be a limitation of gene therapy for this application.

Research Ethics Recommendations for Whole-Genome Research: Consensus Statement

Interest in whole-genome research has grown substantially over the past few months. This article explores the challenging ethics issues associated with this work.

Long-term outcome of fetal cell transplantation on postinfarction ventricular remodeling and function

OBJECTIVES The purpose of this study was to determine the long-term outcome of fetal cell transplantation into myocardial infarction on left ventricular (LV) function and remodeling. BACKGROUND While neonatal cell transplantation improved function for acute myocardial infarction, long-term data on the effects of cell-transplant therapy using a more primitive cell on ventricular remodeling and function are needed.Methods. - Therefore, we injected 4 x 10(6) Fischer 344 fetal cardiac cells or medium into 1-week old infarcts in adult female Fischer rats to assess long-term outcome. RESULTS Ten months after transplantation histologic analysis showed that cell implants were readily visible within the infarct scar. Infarct wall thickness was greater in cell-treated at 0.69 +/- 0.05 mm (n = 11) vs. medium-treated hearts at 0.33 +/- 0.01 mm (n = 19; P = 0.0001). Postmortem LV volume was 0.41 +/- 0.04 ml in cell-treated vs. 0.51 +/- 0.03 ml in medium-treated hearts (P < 0.04). Ejection fraction assessed by LV angiography was 0.40 +/- 0.02 in cell-treated (n = 16) vs. 0.33 +/- 0.02 in medium-treated hearts (n = 24; P < 0.03) with trends towards smaller in vivo end-diastolic and end-systolic volumes in cell-treated vs. medium-treated hearts. Polymerase chain reaction analysis of the Sry gene of the Y chromosome was positive in four of five cell-treated and zero of five medium-treated hearts confirming viability of male cells in female donors. CONCLUSION Over the course of 10 months, fetal cardiac cell transplantation into infarcted hearts increased infarct wall thickness, reduced LV dilatation, and improved LV ejection fraction. Thus, fetal cell-transplant therapy mitigated the longer-term adverse effects of LV remodeling following a myocardial infarction.

Myocardial regeneration: present and future trends.

Cardiomyocytes are terminally differentiated and are unable to proliferate in response to injury. Genetic modulation, cell transplantation and tissue engineering promise a revolutionary approach for myocardial regeneration and tissue repair after myocardial injury. Current data derived from animal models suggest that it may be possible to treat heart failure by inserting genetic materials or myogenic cells into injured myocardium. Success with animal models has raised the hope for new treatment after heart attacks and could prove an alternative to transplantation, particularly in elderly patients for whom there is often a lack of donor hearts. This exciting research, however, still faces significant difficulties before it can develop into a clinical therapeutic tool and many challenges need to be overcome before cell transplantation, gene therapy and tissue engineering can be considered efficient, therapeutic strategies for myocardial regeneration.

Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements.

The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.

Doxorubicin-induced Id2A gene transcription is targeted at an activating transcription factor/cyclic AMP response element motif through novel mechanisms involving protein kinases distinct from protein kinase C and protein kinase A.

We have recently shown that doxorubicin (Dox), an antineoplastic drug and an inhibitor of terminal differentiation of myogenic and adipogenic cells, induces expression of Id, a gene encoding a helix-loop-helix transcriptional inhibitor. In this study we have investigated the molecular mechanisms underlying Dox-induced Id2A expression. We have also attempted to determine whether the genetic responses to Dox are related to the UV response, a well-characterized set of reactions to UV and DNA-damaging compounds that is partly mediated by AP-1. Transient transfection of a series of deletions and point mutation derivatives of the human Id2A promoter sequence shows that two closely spaced and inverted short elements similar to an activating transcription factor (ATF) binding site or a cyclic AMP response element (CRE) are necessary and sufficient for a full response to Dox. We refer to this element as the IdATF site. Sequences containing an IdATF site conferred Dox inducibility on a minimal heterologous promoter. An electrophoretic mobility shift assay showed nuclear proteins specifically interacting with the IdATF sequence. While oligonucleotides containing either legitimate ATF/CRE or AP-1 binding sequences competed for binding, antibody supershift experiments suggested that neither CREB/ATF-1 nor AP-1 are major factors binding to IdATF. Several independent criteria suggest that Dox inducibility was independent of Ca2+/phospholipid-dependent protein kinase (protein kinase C), cyclic AMP-dependent protein kinase (protein kinase A), and tyrosine kinase. Moreover, we found that Dox also induces transcription from promoters of immediate-early genes through an AP-1-independent pathway. Taken together, our results suggest that Dox elicits a novel genetic response distinct from the classical UV response.

Neural tropomodulin: developmental expression and effect of seizure activity

Tropomodulin is a 40.6 kDa tropomyosin-binding protein associated with actin filaments in muscle and the membrane cytoskeleton in erythrocytes. We have detected tropomodulin mRNA and protein in brains of rats by northern and western blot analyses. In situ hybridization of rat brain and spinal cord sections shows tropomodulin expression in the cerebellum, neocortex, hippocampus, and anterior horn of the spinal cord. Tropomodulin expression is first observed around day 15 after birth and increases through day 24. The temporal and spatial changes in tropomodulin expression during cerebellar development parallel those for brain tropomyosin. Tropomodulin mRNA increases in the dentate gyrus of the hippocampus following prolonged seizure activity induced by kainic acid administration; the increase is clearly evident 8 h after initiation of seizures and is still present 1 week later. However, Western blot analysis of tropomodulin protein level in the dentate gyrus before and after seizure induction show only slight increases in tropomodulin protein concentration, suggesting tight regulation of tropomodulin expression at the translational level. The developmental expression of tropomodulin, together with the induction of tropomodulin mRNA production in the dentate gyrus after kainic acid treatment, suggests a role for tropomodulin in neuronal organization and plasticity.

Tropomodulin in rat cardiac muscle. Localization of protein is independent of messenger RNA distribution during myofibrillar development.

Tropomodulin is a 40.6-kD protein that colocalizes with actin filament pointed ends in skeletal muscle. We report the sequence of two partial-length complementary DNA (cDNA) clones of rat cardiac tropomodulin that cover 90% of the coding region. The cDNA sequence is 90% conserved between human and rat, with the predicted amino acid sequence similarity even higher at 95%. Anti-tropomodulin antibodies label a single polypeptide with an apparent mobility of 43,000 in Western blot analysis of rat cardiac muscle. Immunofluorescence experiments using this anti-tropomodulin antibody result in labeling that is coincident with thin filament ends, as demonstrated by double localization with alpha-actinin antibody. Tropomodulin protein is organized into a sarcomeric staining pattern with the earliest appearance of myofibrils in rat cardiocytes. The localization of tropomodulin protein at or near thin filament ends led us to examine the distribution of tropomodulin messenger RNA (mRNA) during myofibrillar development in vitro. Fluorescent in situ hybridization experiments using tropomodulin cDNA probe in cardiocytes that have been cultured for 3 to 5 days show a distribution of large mRNA patches. The cytoplasmic location of tropomodulin mRNA at this time, which bears no relation to the developed myofibrils, suggests that tropomodulin protein is targeted to thin filament ends rather than using localized translational machinery. However, the distribution of tropomodulin mRNA in cultured cardiocytes changes over the next 2 weeks from large perinuclear patches to small concentrations arranged along myofibrils throughout the cell. The reorganization of tropomodulin mRNA throughout the cardiocyte appears to be distinct from the pattern of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same time period. Increasing intracellular density of myofibrils within developing cardiocytes may lead to redistribution of selected mRNAs for localized translation.