Niladri Mal

SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction

Stem cell transplantation at the time of acute myocardial infarction (AMI) improves cardiac function. Whether the improved cardiac function results from regeneration of cardiac myocytes, modulation of remodeling, or preservation of injured tissue through paracrine mechanisms is actively debated. Because no specific stem cell population has been shown to be optimal, we investigated whether the benefit of stem cell transplantation could be attributed to a trophic effect on injured myocardium. Mesenchymal stem cells secrete SDF‐1 and the interaction of SDF‐1 with its receptor, CXCR4, increases survival of progenitor cells. Therefore, we compared the effects of MSC and MSC engineered to overexpress SDF‐1 on cardiac function after AMI. Tail vein infusion of syngeneic MSC and MSC:SDF‐1 1 day after AMI in the Lewis rat led to improved cardiac function by echocardiography by 70.7% and 238.8%, respectively, compared with saline controls 5 wk later. The beneficial effects of MSC and MSC:SDF‐1 transplantation were mediated primarily through preservation, not regeneration of cardiac myocytes within the infarct zone. The direct effect of SDF‐1 on cardiac myocytes was due to the observation that’ between 24 and 48 h after AMI, SDF‐1‐expressing MSC increased cardiac myocyte surviva, vascular density (18.2±4.0 vs. 7.6±2.3 vessels/mm2, P<0.01; SDF‐1:MSC vs. MSC), and cardiac myosin‐positive area (MSC: 49.5%;mSC:SDF‐1: 162.1%) within the infarct zone. There was no evidence of cardiac regeneration by the infused MSC or endogenous cardiac stem cells based on lack of evidence for cardiac myocytes being derived from replicating cells. These results indicate that stem cell transplantation may have significant beneficial effects on injured organ function independent of tissue regeneration and identify SDF‐1:CXCR4 binding as a novel target for myocardial preservation.—Zhang, M., Mal, N., Kiedrowski, M., Chacko, M., Askari, A. T., Popovic, Z. B., Koc, O. N., Penn, M. S. SDF‐1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction. FASEB J. 21, 3197–3207 (2007)

Monocyte Chemotactic Protein‐3 Is a Myocardial Mesenchymal Stem Cell Homing Factor

MSCs have received attention for their therapeutic potential in a number of disease states, including bone formation, diabetes, stem cell engraftment after marrow transplantation, graft‐versus‐host disease, and heart failure. Despite this diverse interest, the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors, we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts, which do not home after MI. This analysis identified monocyte chemotactic protein‐3 (MCP‐3) as a potential MSC homing factor. Overexpression of MCP‐3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs, cardiac function improved in MCP‐3‐expressing hearts (88.7%, p < .001) but not in control hearts (8.6%, p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather, MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP‐3 is an MSC homing factor; local overexpression of MCP‐3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration.

SDF-1 recruits cardiac stem cell-like cells that depolarize in vivo.

Prolongation or reestablishment of stem cell homing through the expression of SDF-1 in the myocardium has been shown to lead to homing of endothelial progenitor cells to the infarct zone with a subsequent increase in vascular density and cardiac function. While the increase in vascular density is important, there could clearly be other mechanisms involved. In a recent study we demonstrated that the infusion of mesenchymal stem cells (MSC) and MSC that were engineered to overexpress SDF-1 led to significant decreases in cardiac myocyte apoptosis and increases in vascular density and cardiac function compared to control. In that study there was no evidence of cardiac regeneration from either endogenous stem cells or the infused mesenchymal stem cells. In this study we performed further detailed immunohistochemistry on these tissues and demonstrate that the overexpression of SDF-1 in the newly infracted myocardium led to recruitment of small cardiac myosin-expressing cells that had proliferated within 2 weeks of acute MI. These cells did not differentiate into mature cardiac myocytes, at least by 5 weeks after acute MI. However, based on optical mapping studies, these cells appear capable of depolarizing. We observed greater optical action potential amplitude in the infarct border in those animals that received SDF-1 overexpressing MSC than observed in noninfarcted animals and those that received control MSC. Further immunohistochemistry revealed that these proliferated cardiac myosin-positive cells did not express connexin 43, but did express connexin 45. In summary, our study suggests that the prolongation of SDF-1 expression at the time of acute MI leads to the recruitment of endogenous cardiac myosin stem cells that may represent cardiac stem cells. These cells are capable of depolarizing and thus may contribute to increased contractile function even in the absence of maturation into a mature cardiac myocyte.

Direct delivery of syngeneic and allogeneic large-scale expanded multipotent adult progenitor cells improves cardiac function after myocardial infarct

BACKGROUND Multipotent adult progenitor cells (MAPC) comprise interesting candidates for myocardial regeneration because of a broad differentiation ability and immune privilege. We aimed to compare the improvement of cardiac function by syngeneic and allogeneic MAPC produced on a large scale using a platform optimized from MAPC research protocols. METHODS Myocardial infarction was induced in Lewis rats by direct left anterior descending ligation followed immediately by direct injection into the infarct border zone of either Sprague-Dawley or Lewis MAPC from large-scale expansions. Echocardiography was performed to evaluate improvement in cardiac function, and immunohistochemistry was performed to identify MAPC within the infarct zone. RESULTS Significant increases were observed in functional performance in animals transplanted with expanded MAPC compared with saline controls, with no significant differences between the syngeneic and allogeneic groups. Immunostaining demonstrated significant engraftment of expanded MAPC at 1 day after acute myocardial infarction, with <10% of either syngeneic or allogeneic cells remaining at 6 weeks. At this point there was no evidence of myocardial regeneration. However, a significant increase in vascular density within the infarct zone in MAPC-transplanted animals was observed, and MAPC were found to produce high levels of VEGF in culture. DISCUSSION These findings support a model in which delivery of expanded MAPC following acute myocardial infarction results in improvement in cardiac function because of paracrine effects resulting in vascular density increases, as well as potentially other trophic effects, supporting newly injured cardiac myocytes. Thus transplantation with MAPC may represent a promising therapeutic strategy with application in the stimulation of neovascularization in ischemic heart disease.

Disruption of Protein Kinase A Interaction with A-kinase-anchoring Proteins in the Heart in Vivo EFFECTS ON CARDIAC CONTRACTILITY, PROTEIN KINASE A PHOSPHORYLATION, AND TROPONIN I PROTEOLYSIS

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.

Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis

Superparamagnetic iron oxide (SPIO) particles have been used successfully as an intracellular contrast agent for nuclear MRI cell tracking in vivo. We present a method of detecting intracellular SPIO colloid uptake in live cells using cell magnetophoresis, with potential applications in measuring intracellular MRI con trast uptake. The method was evaluated by measuring shifts in mean and distribution of the cell magnetophoretic mobility, and the concomitant changes in popu lation frequency of the magnetically positive cells when compared to the unmanipulated negative control. Seven different transfection agent (TA) ‐SPIO complexes based on dendrimer, lipid, and polyethylenimine compounds were used as test standards, in combination with 3 differ ent cell types: mesenchymal stem cells, cardiac fibroblasts, and cultured KG‐1a hematopoietic stem cells. Transfectol (TRA) ‐SPIO incubation resulted in the highest frequency of magnetically positive cells (>90%), and Fugene 6 (FUG) ‐SPIO incubation the lowest, below that when using SPIO alone. A highly regular process of cell mag netophoresis was amenable to intracellular iron mass calculations. The results were consistent in all the cell types studied and with other reports. The cell magnetophoresis depends on the presence of high‐spin iron species and is therefore expected to be directly related to the cell MRI contrast level.— Jing, Y., Mal, N., Williams, P. S., Mayorga, M., Penn, M. S., Chalmers, J. J., Zborowski, M. Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis. FASEB J. 22, 4239–4247 (2008)

Platelet, Not Endothelial, P-Selectin Is Required for Neointimal Formation After Vascular Injury

Background—P-selectin blockade significantly inhibits inflammation and neointimal formation after arterial injury; however, the independent roles of platelet and endothelial P-selectins in this process are unknown. In atherosclerosis, both platelet and endothelial cell P-selectins are important. This study was designed to determine whether P-selectin expression on platelet, endothelial, or both surfaces is critical to the inflammatory response and neointimal formation after arterial injury. Methods and Results—Using wild-type (WT) and P-selectin–knockout (Psel−/−) mice, we performed bone marrow transplantation to generate chimeric mice that expressed either platelet P-selectin (Plt-Psel) or endothelial P-selectin (EC-Psel). Double injury of the carotid artery was performed in these mice as well as in WT and Psel−/− mice. Animals were euthanized 4 or 21 days after arterial injury. Morphometric data showed that there was more neointimal formation in the WT mouse group when compared with the Psel−/− mouse group (0.015±0.004 vs 0.004±0.004 mm2, P<0.001). Further comparison showed significantly less neointimal area in EC-Psel mice (0.006±0.004 mm2) compared with Plt-Psel mice (0.011±0.005 mm2, P=0.026) and WT mice (0.015±0.004 mm2, P=0.001). No significant differences were observed between WT and Plt-Psel mice or between Psel−/− and EC-Psel mice. Decreased neointimal formation was accompanied by a reduced inflammatory response, as evidenced by immunostaining of RANTES and MCP-1 4 days after injury. Conclusions—Platelet P-selectin expression, but not endothelial P-selectin, plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting leukocyte-platelet interactions could be effective in inhibiting restenosis.

Engineered cell therapy for sustained local myocardial delivery of nonsecreted proteins.

Novel strategies for the treatment of congestive heart failure have taken the form of gene and cell therapy to induce angiogenesis, optimize calcium handling by cardiac myocytes, or regenerate damaged myocardial tissue. Arguably both gene- and cell-based therapies would be benefited by having the ability to locally deliver specific transcription factors and other usually nonsecreted proteins to cells in the surrounding myocardial tissue. The herpes simplex virus type 1 (HSV-1) tegument protein VP22 has been shown to mediate protein intercellular trafficking to mammalian cells and finally localize into the nucleus, which makes it a useful cargo-carrying functional protein in cell-based gene therapy. While VP22 has been studied as a means to modulate tumor growth, little is known about the distribution and transport kinetics of VP22 in the heart and its potential application in combination with autologous cell transplantation for the delivery of proteins to myocardial tissue. The aim of this study was to evaluate the efficacy of VP22 fusion protein intercellular trafficking combined with autologous cell transplantation in the heart. In an in vitro study untransfected rat heart cells were cocultured with stably transfected rat cardiac fibroblasts (RCF) with fusion constructs of VP22. The control experiment was untransfected rat heart cells co-plated with RCF stably transfected with enhanced green fluorescence protein (eGFP). The Lewis rat model was selected for in vivo study. In the in vitro studies there was a 14-fold increase in the number of GFP-positive cells 48 h after initiating coculture with VP22-eGFP RCF compared to eGFP RCF. In the rat model, transplantation of VP22-eGFP expressing RCF led to VP22-eGFP fusion protein delivery to an area of myocardial tissue that was 20-fold greater than that observed when eGFP RCF were transplanted. This area appeared to reach a steady state between 7 and 10 days after transplantation. The VP22-eGFP area consisted of eGFP-positive endothelium, smooth muscle cells, and cardiac myocytes with delivery to an area of approximately 1 mm2 of myocardial tissue. Our data suggest a viable strategy for the delivery of proteins that are not naturally secreted or internalized, and provide the first insight into the feasibility and effectiveness of cell-penetrating proteins combined with cell transplantation in the heart.

Stem cells in cardiovascular disease: methods and protocols.

Stem cells are cells capable of proliferation, self-renewal, and differentiation into various organ-specific cell types. Stem cells are subclassified based on their species of origin (mice, rat, human), developmental stage of the species (embryonic, fetal, or adult), tissue of origin (hematopoietic, mesenchymal, skeletal, neural), and potential to differentiate into one or more specific types of mature cells (totipotent, pluripotent, multipotent). Embryonic stem (ES) cells are totipotent, primitive cells derived from the embryo that have the potential to become all specialized cell types. Conversely, adult stem cells are undifferentiated cells found in differentiated tissue that retain the potential to renew themselves and differentiate to yield organ-specific tissues. Stem cells are attractive candidates for novel therapeutics for patients with different heart diseases, including congestive heart failure, most commonly caused by myocardial infarction. The remarkable proliferative and differentiation capacity of stem cells promises an almost unlimited supply of specific cell types including viable functioning cardiomyocytes to replace the scarred myocardium following transplantation.

Multipotent Adult Progenitor Cells

In 2001 the laboratory of Catherine Verfaillie at the University of Minnesota described the multipotent adult progenitor cell (MAPC) as a novel progenitor cell present in adult marrow that is biologically and antigenically distinct from the mesenchymal stem cell (MSC). MAPCs represent a more primitive progenitor cell population than MSCs and demonstrate remarkable differentiation capability along the epithelial, endothelial, neuronal, myogenic, hematopoeitic, osteogenic, hepatic, chondrogenic, and adipogenic lineages. MAPCs thus embody a unique class of adult stem cells that emulate the broad biological plasticity characteristic of embryonic stem (ES) cells, while maintaining the characteristics that make adult stem cells more amenable to therapeutic application. MAPCs have been reported to be capable of prolonged culture without loss of differentiation potential, and of showing efficient, long-term engraftment and differentiation along multiple developmental lineages in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice without evidence of teratoma formation. Based on these findings, there is great interest in evaluating the therapeutic value of MAPCs for a variety of human genetic and degenerative ailments, including cardiovascular disease.