Therapeutic effects of human STRO-3-selected mesenchymal precursor cells and their soluble factors in experimental myocardial ischemia.

Stromal precursor antigen (STRO)-3 has previously been shown to identify a subset of adult human bone marrow (BM)-derived mesenchymal lineage precursors, which may have cardioprotective potential. We sought to characterize STRO-3-immunoselected and culture-expanded mesenchymal precursor cells (MPCs) with respect to their biology and therapeutic potential in myocardial ischemia. Immunoselection of STRO-3(+) MPCs enriched for fibroblastic colony forming units from unfractionated BM mononuclear cells (MNCs). Compared to mesenchymal stem cells conventionally isolated by plastic adherence, MPCs demonstrated increased proliferative capacity during culture expansion, expressed higher levels of early 'stem cell' markers and various pro-angiogenic and cardioprotective cytokines, and exhibited greater trilineage developmental efficiency. Intramyocardial injection of MPCs into a rat model of myocardial infarction (MI) promoted left ventricular recovery and inhibited left ventricular dilatation. These beneficial effects were associated with cardioprotective and pro-angiogenic effects at the tissue level, despite poor engraftment of cells. Treatment of MI rats with MPC-conditioned medium (CM) preserved left ventricular function and dimensions, reduced myocyte apoptosis and fibrosis, and augmented neovascularization, involving both resident vascular cells and circulating endothelial progenitor cells (EPCs). Profiling of CM revealed various cardioprotective and pro-angiogenic factors, which had biological activity in cultures of myocytes, tissue-resident vascular cells and EPCs. Prospective immunoselection of STRO-3(+) MPCs from BM MNCs conferred advantage in maintaining a population of immature MPCs during ex vivo expansion. Transplantation of culture-expanded MPCs into the post-MI heart resulted in therapeutic benefit, attributable at least in part to paracrine mechanisms of action. Thus, MPCs represent a promising therapy for myocardial ischemia.

Mesenchymal cell transplantation and myocardial remodeling after myocardial infarction.

BACKGROUND: Targeted delivery of mesenchymal precursor cells (MPCs) can modify left ventricular (LV) cellular and extracellular remodeling after myocardial infarction (MI). However, whether and to what degree LV remodeling may be affected by MPC injection post-MI, and whether these effects are concentration-dependent, remain unknown. METHODS AND RESULTS: Allogeneic MPCs were expanded from sheep bone marrow, and direct intramyocardial injection was performed within the borderzone region 1 hour after MI induction (coronary ligation) in sheep at the following concentrations: 25x10(6) (25 M, n=7), 75x10(6) (75 M, n=7), 225x10(6) (225 M, n=10), 450x10(6) (450 M, n=8), and MPC free media only (MI Only, n=14). LV end diastolic volume increased in all groups but was attenuated in the 25 and 75 M groups. Collagen content within the borderzone region was increased in the MI Only, 225, and 450 M groups, whereas plasma ICTP, an index of collagen degradation, was highest in the 25 M group. Within the borderzone region matrix metalloproteinases (MMPs) and MMP tissue inhibitors (TIMPs) also changed in a MPC concentration-dependent manner. For example, borderzone levels of MMP-9 were highest in the 25 M group when compared to the MI Only and other MPC treatment group values. CONCLUSIONS: MPC injection altered collagen dynamics, MMP, and TIMP levels in a concentration-dependent manner, and thereby influenced indices of post-MI LV remodeling. However, the greatest effects with respect to post-MI remodeling were identified at lower MPC concentrations, thus suggesting a therapeutic threshold exists for this particular cell therapy.

Allogeneic mesenchymal precursor cell therapy to limit remodeling after myocardial infarction: the effect of cell dosage.

BACKGROUND: This experiment assessed the dose-dependent effect of a unique allogeneic STRO-3-positive mesenchymal precursor cell (MPC) on postinfarction left ventricular (LV) remodeling. The MPCs were administered in a manner that would simulate an off-the-self, early postinfarction, preventative approach to cardiac cell therapy in a sheep transmural myocardial infarct (MI) model. METHODS: Allogeneic MPCs were isolated from male crossbred sheep. Forty-six female sheep underwent coronary ligation to produce a transmural LV anteroapical infarction. One hour after infarction, the borderzone myocardium received an injection of 25, 75, 225, or 450 x 10(6) MPCs, or cell medium. Echocardiography was performed at 4 and 8 weeks after MI to quantify LV end-diastolic (LVEDV) and end-systolic volumes (LVESV), ejection fraction (EF), and infarct expansion. CD31 and smooth muscle actin (SMA) immunohistochemical staining was performed on infarct and borderzone specimens to quantify vascular density. RESULTS: Compared with controls, low-dose (25 and 75 x 10(6) cells) MPC treatment significantly attenuated infarct expansion and increases in LVEDV and LVESV. EF was improved at all cell doses. CD31 and SMA immunohistochemical staining demonstrated increased vascular density in the borderzone only at the lower cell doses. There was no evidence of myocardial regeneration within the infarct. CONCLUSION: Allogeneic STRO-3 positive MPCs attenuate the remodeling response to transmural MI in a clinically relevant large-animal model. This effect is associated with vasculogenesis and arteriogenesis within the borderzone and infarct and is most pronounced at lower cell doses.

Células-tronco mesenquimais para reparo clínico / Allogeneic mesenchymal stem cells for cardiac repair

As células tronco mesenquimais (CTMs) são uma população adulta de células que compartilham algumas características fenotípicas com as células-tronco embrionárias, mas carecem da preocupação ética ou da segurança associadas a tais células indiferenciadas. As CTMs estão localizadas, principalemnte na médula óssea e, em menor grau, em outros tecidos. Elas podem ser positivamente selecionadas, expandidas em cultura ex vivo e se diferenciar para a linhagem de osteócitos, condrócitos e adipócitos do tecido mesenquimal. As chamadas células mesenquimais progenitoras (CMPs), precusoras de CTMs, podem ser isoladas da medula óssea, resultando em produto homogêneo, contendo uma população marcadamente mais pura de CTMs, ou seja, sem o elevado número de células contaminantes...

Mesenchymal stem cells (MSCs) are an adult stem cell population that share some phenotypic characteristics with embryonic stem cells but lack the ethical or safety concerns associated with such undifferentiated cells. MSCs are located primarily in the bone marrow and to a lesser extent other tissues and can be positively selected, ex vivo culture expanded and are able to differentiate into the mesenchymal tissue lineage of osteocytes, chondrocytes and adipocytes. A precursor of MSCs termed mesenchymal progenitor cells (MPCs) can be further isolated from bone marrow resulting in a homogenous product containing a significantly purer population of MSCs without the high number of contaminating cells inherent toMSC isolation techniques. Additionally, MSCs lack certain co-stimulatory receptors such as HLA class II and locally secrete factors downregulating T cell responses allowing for allogeneic usage as an off-the-shelf product. MSCs increase neovascularization and cardiomyocyte regeneration in a number of animal models resulting in improvement in cardiac functional outcome and limitation of the progression to heart failure. The mechanism(s) of improvement remains a matter of debate. Alternatives include direct differentiation of the MSCs vs. paracrine secretion of factors that indirectly effect endogenous tissue and local progenitor cells. While the exact mechanism of cardiac improvement continues to be explored, it is clear that allogeneic MSCs offer a reproducible, inexpensive, regulatory-friendly cellular therapy treatment for cardiovascular disease that will need to be investigated in larger safety and efficacy clinical trials.

Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium.

Mesenchymal lineage precursors can be reproducibly isolated from adult mammalian bone marrow and grown in culture. Immunoselection with monoclonal antibodies against STRO-1 and vascular-cell-adhesion molecule 1 (VCAM1/CD106) prior to expansion results in a 1,000-fold enrichment of mesenchymal precursors compared to standard isolation techniques. Intramyocardial injection of human STRO-1-selected precursors in an athymic rat model of acute myocardial infarction results in induction of vascular network formation and arteriogenesis coupled with global functional cardiac recovery.

Catalytic degradation of vitamin D up-regulated protein 1 mRNA enhances cardiomyocyte survival and prevents left ventricular remodeling after myocardial ischemia.

Vitamin D3 up-regulated protein 1 (VDUP1) is a key mediator of oxidative stress on various cellular processes via downstream effects on apoptosis signaling kinase 1 (ASK1) and p38 mitogen-activated protein kinase (MAPK). Here, we report that VDUP1 expression is significantly increased in rat hearts following acute myocardial ischemia, suggesting it may have important regulatory effects on cardiac physiological processes during periods of oxidative stress. Transfection of H9C2 cardiomyoblasts with a sequence-specific VDUP1 DNA enzyme to down-regulate VDUP1 mRNA expression significantly reduced apoptosis and enhanced cell survival under conditions of H(2)O(2) stress, and these effects involved inhibition of ASK1 activity. Direct intracardiac injection of the DNA enzyme at the time of acute myocardial infarction reduced myocardial VDUP1 mRNA expression and resulted in prolonged reduction in cardiomyocyte apoptosis and ASK1 activity. Moreover, down-regulation of VDUP1 was accompanied by significant reduction in cardiac expression of pro-collagen type I alpha2 mRNA level, as well as marked reduction in myocardial scar formation. These features were accompanied by significant improvement in cardiac function. Together, these results suggest a direct role for VDUP1 in the adverse effects of ischemia and oxidative stress on cardiomyocyte survival, left ventricular collagen deposition, and cardiac function. Strategies to inhibit VDUP1 expression and/or function during acute ischemic events may be beneficial to cardiac functional recovery and prevention of left ventricular remodeling.

Downregulated expression of plasminogen activator inhibitor-1 augments myocardial neovascularization and reduces cardiomyocyte apoptosis after acute myocardial infarction.

OBJECTIVES: The aim of this study was to examine whether selective plasminogen activator inhibitor type 1 (PAI-1) downregulation in the acutely ischemic heart increases the myocardial microvasculature and improves cardiomyocyte (CM) survival. BACKGROUND: Endogenous myocardial neovascularization is an important process enabling cardiac functional recovery after acute myocardial infarction. Expression of PAI-1, a potent inhibitor of angiogenesis, is induced in ischemic heart tissue. METHODS: A sequence-specific catalytic deoxyribonucleic acid (DNA) enzyme was used to reduce PAI-1 levels in cultured endothelial cells and in ischemic myocardium. At the time of coronary artery ligation, rats were randomized into three groups, each receiving an intramyocardial injection (IMI) of a single dose at three different sites of the peri-infarct region consisting, respectively, of DNA enzyme E2 targeting rat PAI-1 (E2), scrambled control DNA enzyme (E0), or saline. Cardiomyocyte apoptosis, capillary density, and echocardiography were studied two weeks following infarction. RESULTS: The E2 DNA enzyme, which efficiently inhibited rat PAI-1 expression in vitro, induced prolonged suppression (>2 weeks) of PAI-1 messenger ribonucleic acid and protein in rat heart tissues after a single IMI. At two weeks, hearts from experimental rats had over five-fold greater capillary density, 70% reduction in apoptotic CMs, and four-fold greater functional recovery compared with controls. CONCLUSIONS: These results imply a causal relationship between elevated PAI-1 levels in ischemic hearts and adverse outcomes, and they suggest that strategies to reduce cardiac PAI-1 activity may augment neovascularization and improve functional recovery.

Down-regulation of plasminogen activator inhibitor 1 expression promotes myocardial neovascularization by bone marrow progenitors.

Human adult bone marrow-derived endothelial progenitors, or angioblasts, induce neovascularization of infarcted myocardium via mechanisms involving both cell surface urokinase-type plasminogen activator, and interactions between beta integrins and tissue vitronectin. Because each of these processes is regulated by plasminogen activator inhibitor (PAI)-1, we selectively down-regulated PAI-1 mRNA in the adult heart to examine the effects on postinfarct neovascularization and myocardial function. Sequence-specific catalytic DNA enzymes inhibited rat PAI-1 mRNA and protein expression in peri-infarct endothelium within 48 h of administration, and maintained down-regulation for at least 2 wk. PAI-1 inhibition enhanced vitronectin-dependent transendothelial migration of human bone marrow-derived CD34+ cells, and resulted in a striking augmentation of angioblast-dependent neovascularization. Development of large, thin-walled vessels at the peri-infarct region was accompanied by induction of proliferation and regeneration of endogenous cardiomyocytes and functional cardiac recovery. These results identify a causal relationship between elevated PAI-1 levels and poor outcome in patients with myocardial infarction through mechanisms that directly inhibit bone marrow-dependent neovascularization. Strategies that reduce myocardial PAI-1 expression appear capable of enhancing cardiac neovascularization, regeneration, and functional recovery after ischemic insult.

Myocardial neovascularization by adult bone marrow-derived angioblasts: strategies for improvement of cardiomyocyte function.

In the pre-natal period, hemangioblasts derived from the human ventral aorta give rise to cellular elements involved in both hematopoiesis and vasculogenesis, resulting in formation of the primitive capillary network. Endothelial precursors with phenotypic and functional characteristics of embryonic hemangioblasts are also present in human adult bone marrow, and can be used to induce infarct bed vasculogenesis and angiogenesis after experimental myocardial infarction. The neovascularization results in decreased apoptosis of hypertrophied myocytes in the peri-infarct region, long-term salvage and survival of viable myocardium, reduction in collagen deposition, and sustained improvement in cardiac function. Autologous angioblasts may also be useful in cellular therapy strategies aiming to regenerate myocardial tissue after established heart failure. It is likely that protocols using cardiomyocyte/mesenchymal stem cells will require balanced co-administration of angioblasts to provide vascular structures for supply of oxygen and nutrients to both the chronically ischemic, endogenous myocardium and to the newly-implanted cardiomyocytes. Future studies will need to address the timing, relative concentrations, source and route of delivery of each of these cellular populations in animal models of acute and chronic myocardial ischemia.

New directions in strategies using cell therapy for heart disease.

Congestive heart failure remains a major public health problem and is frequently the end result of cardiomyocyte apoptosis and fibrous replacement after myocardial infarction, a process referred to as left ventricular remodeling. Cardiomyocytes undergo terminal differentiation soon after birth and are generally considered to irreversibly withdraw from the cell cycle. In response to ischemic insult adult cardiomyocytes undergo cellular hypertrophy, nuclear ploidy, and a high degree of apoptosis. A small number of human cardiomyocytes retain the capacity to proliferate and regenerate in response to ischemic injury. However, whether these cells are derived from a resident pool of cardiomyocyte stem cells or from a renewable source of circulating bone marrow-derived stem cells that home to the damaged myocardium is at present not known. Replacement and regeneration of functional cardiac muscle after an ischemic insult to the heart could be achieved by either stimulating proliferation of endogenous mature cardiomyocytes or resident cardiac stem cells or by implanting exogenous donor-derived or allogeneic cells such as fetal or embryonic cardiomyocyte precursors, bone marrow derived mesenchymal stem cells, or skeletal myoblasts. The newly formed cardiomyocytes must integrate precisely into the existing myocardial wall in order to augment synchronized contractility and avoid potentially life-threatening alterations in the electrical conduction of the heart. A major impediment to survival of the implanted cells is altered immunogenicity by prolonged ex vivo culture conditions. In addition, concurrent myocardial revascularization is required to ensure viability of the repaired region and prevent further scar tissue formation. Human adult bone marrow contains endothelial precursors which resemble embryonic angioblasts and can be used to induce infarct bed neovascularization after experimental myocardial infarction. This results in protection of cardiomyocytes against apoptosis, induction of cardiomyocyte proliferation and regeneration, long-term salvage and survival of viable myocardium, prevention of left ventricular remodeling, and sustained improvement in cardiac function. It is reasonable to anticipate that cell therapy strategies for ischemic heart disease will need to incorporate (a) a renewable source of proliferating, functional cardiomyocytes, and (b) angioblasts to generate a network of capillaries and larger size blood vessels for supply of oxygen and nutrients to both the chronically ischemic endogenous myocardium and to the newly implanted cardiomyocytes