Clonality of mouse and human cardiomyogenesis in vivo.

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.

Formation of large coronary arteries by cardiac progenitor cells.

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kit-positive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1alpha, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.

Expression and regulation of type 2 iodothyronine deiodinase in rat aorta media.

Here, we have found that type 2 iodothyronine deiodinase (D2) is present in rat aorta media and that there is a circadian variation in the D2 expression. The D2 mRNA was approximately 4-fold higher at 0900 h than at 2100 h, and the activity was approximately 6-fold higher at noon than at 2100 h. The increase in aorta media D2 activity is preceded by the increase in its mRNA. The increase in D2 mRNA and activity in the circadian variation was reduced by the administration of prazosin, an alpha1-adrenergic antagonist, and propranolol, a beta- adrenergic antagonist. Furthermore, phenylephrine, an alpha1-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, caused a significant increase in D2 mRNA and activity. In the hypothyroid rats, aorta mediae D2 mRNA at both 0900 and 2100 h were not significantly different when compared with those in the euthyroid rats. On the other hand, aorta mediae D2 activity at both 1200 and 2100 h in the hypothyroid rats were approximately 2-fold higher. From these results, we suggest that D2 activity of rat aorta media is increased by both alpha1- and beta-adrenergic stimulation, at least partly, at the pretranslational level. We also suggest that both alpha1- and beta-adrenergic mechanisms may be involved, at least partly, in the circadian variation of the activity. In the hypothyroid state, the aorta media D2 activity is increased mainly by the posttranslational mechanism, and the similar circadian variation of the D2 expression is present as in the euthyroid state.

Type 2 deiodinase expression is stimulated by growth factors in human vascular smooth muscle cells.

Type 2 deiodinase (D2) catalyzes the conversion of the prohormone T4 to the biologically active T3. D2 is expressed in human aortic smooth muscle cells (hASMCs). In this study, we demonstrated that the D2 mRNA and activity in hASMCs were up-regulated by platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF). The induction of D2 mRNA by PDGF-BB and bFGF was dependent on de novo RNA and protein synthesis. PD98059, a specific inhibitor of the upstream kinase that activates extracellular signal-regulated kinase (ERK), significantly suppressed the induction by both PDGF-BB and bFGF. SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, and SP600125, a specific inhibitor of c-Jun N-terminal kinase (JNK), also reduced the induction by both PDGF-BB and bFGF. These results suggest that both PDGF-BB and bFGF induce D2 expression at least partly via ERK pathway. The p38 MAP kinase and JNK pathways may also be involved in the induction.

Thyroid hormone activation in vascular smooth muscle cells is negatively regulated by glucocorticoid.

BACKGROUND: Type 2 iodothyronine deiodinase (D2) catalyzes the production of triiodothyronine from thyroxine. D2 is present in rat aorta media, and there is a circadian variation in the D2 expression. In rat aorta media, the D2 activity exhibited the maximal value at 1200 hour and low value between 1800 and 2400 hour. To understand the mechanisms that induce the circadian variation in the D2 expression, we examined the effects of glucocorticoid on the D2 activity and mRNA in rat aorta media and cultured vascular smooth muscle cells (VSMCs). METHODS: The effects of intrinsic and extrinsic glucocorticoid on the D2 activity and mRNA in rat aorta media were studied using metyrapone, a corticosterone synthesis inhibitor, and dexamethasone (DEX). Further, the effects of DEX on D2 expression were studied using the cultured rat VSMCs. RESULTS: The trough values of D2 activity and mRNA at 2100 hour were increased by the treatment with metyrapone. On the other hand, the peak values of D2 activity and mRNA were decreased by the treatment with DEX. D2 activity and mRNA in cultured rat VSMCs were increased by the addition of 10(-3) M dibutyryl cyclic adenosine monophosphate [(Bu)(2)cAMP]. The increments were reduced by coincubation with 10(-6) M DEX. CONCLUSIONS: These results suggest that glucocorticoids might directly suppress the D2 expression in rat VSMCs induced by a cAMP-dependent mechanism.

Human cardiac stem cells.

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.