Shear stress-induced mechanotransduction protein deregulation and vasculopathy in a mouse model of progeria.

INTRODUCTION: A mouse model of progeria derived by insertion of the human mutant LMNA gene (mLMNA), producing mutant lamin A, shows loss of smooth muscle cells in the media of the ascending aorta. We hypothesized that high shear stress, in the presence of mutant lamin A, induces this vasculopathy and tried to define the molecular and cellular basis for aortic vasculopathy. METHODS: Ascending and descending aortas from wild type (WT) and mLMNA+ mice were compared using proteomics, Western blots, PCR and immunostaining. To determine whether high fluidic shear stress, known to occur in the ascending aorta, contributed to the vasculopathy, we exposed descending aortas of mLMNA+ mice, with no apparent vasculopathy, to 75 dynes/cm2 shear stress for 30 minutes using a microfluidic system. RESULTS: When the mice were one year of age, expression of several mechanotransduction proteins in the ascending aorta, including vimentin, decreased in mLMNA+ mice but no decrease occurred in the descending aorta. High fluidic shear stress produced a significant reduction in vimentin of mLMNA+ mice but not in similarly treated WT mice. CONCLUSIONS: The occurrence of mutant lamin A and high shear stress correlate with a reduction in the level of mechanotransduction proteins in smooth muscle cells of the media. Reduction of these proteins may contribute over time to development of vasculopathy in the ascending aorta in progeria syndrome.

Cardiomyogenesis in the adult human heart.

RATIONALE: The ability of the human heart to regenerate large quantities of myocytes remains controversial, and the extent of myocyte renewal claimed by different laboratories varies from none to nearly 20% per year. OBJECTIVE: To address this issue, we examined the percentage of myocytes, endothelial cells, and fibroblasts labeled by iododeoxyuridine in postmortem samples obtained from cancer patients who received the thymidine analog for therapeutic purposes. Additionally, the potential contribution of DNA repair, polyploidy, and cell fusion to the measurement of myocyte regeneration was determined. METHODS AND RESULTS: The fraction of myocytes labeled by iododeoxyuridine ranged from 2.5% to 46%, and similar values were found in fibroblasts and endothelial cells. An average 22%, 20%, and 13% new myocytes, fibroblasts, and endothelial cells were generated per year, suggesting that the lifespan of these cells was approximately 4.5, 5, and 8 years, respectively. The newly formed cardiac cells showed a fully differentiated adult phenotype and did not express the senescence-associated protein p16(INK4a). Moreover, measurements by confocal microscopy and flow cytometry documented that the human heart is composed predominantly of myocytes with 2n diploid DNA content and that tetraploid and octaploid nuclei constitute only a small fraction of the parenchymal cell pool. Importantly, DNA repair, ploidy formation, and cell fusion were not implicated in the assessment of myocyte regeneration. CONCLUSIONS: Our findings indicate that the human heart has a significant growth reserve and replaces its myocyte and nonmyocyte compartment several times during the course of life.

Bone marrow-derived cells do not repair endothelium in a mouse model of chronic endothelial cell dysfunction.

AIMS: Bone marrow (BM)-derived endothelial progenitor cells (EPCs) in the circulation replace damaged vascular endothelium. We assessed the hypothesis that a BM transplant from healthy animals would restore normal arterial endothelium and prevent hypertension in young endothelial nitric oxide synthase-deficient (eNOS(-/-)) mice. METHODS AND RESULTS: Radiation or busulfan-induced BM ablation in eNOS(-/-) mice on day 6, day 14, or day 28 was followed by a BM transplant consisting of enhanced green fluorescent protein positive (EGFP(+)) cells from C57BL/6J mice. Peripheral blood cell chimerism was always greater than 85% at 4 months after BM transplant. Molecular assays of heart, kidney, and liver revealed low-level chimerism in all treatment groups, consistent with residual circulating EGFP(+) blood cells. When aorta, coronary, renal, hepatic, and splenic arteries in BM-transplanted eNOS(-/-) mice were examined by confocal microscopy, there were no EGFP- or eNOS-positive endothelial cells detected in these vessels in any of the treatment groups. Likewise, telemetry did not detect any reduction in blood pressure. Thus, no differences were observed in our measurements using several different treatment protocols. CONCLUSION: We found no evidence for BM-derived EPC renewal of endothelium in this eNOS-deficient mouse model of a chronic vascular disease or in wild-type mice during postnatal growth. Hence, renewal of chronic dysfunctional endothelium and endothelial homeostasis may be dependent on resident vascular progenitor cells.

AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates.

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34(+) hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34(+) cells were phenotypically analyzed, marked with Neo(R)-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34(+) cells with Neo(R) gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34(+) cells are in the G(1) phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34(+) cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34(+) cells were better than those obtained using CD34(+) cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34(+) cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Adult bone marrow stem cells regenerate myocardium in ischemic heart disease.

Recent studies have demonstrated the existence of several populations of primitive cells in mouse and human bone marrow that have the capacity, both in vitro and in vivo, to give rise to cells of all three germ layers. Mesenchymal/stromal stem cells and hematopoietic stem cells are the leading candidates for this activity that some believe may recapitulate the potential of embryonic stem cells. Very little is known about the molecular controls for this adult stem cell activity commonly referred to as transdifferentiation or plasticity. Regeneration of a large number of cell types and tissues has been investigated with one of the most extensively studied being the myocardium. These studies involved ligation of the left coronary artery in adult mice and the direct injection or mobilization of bone marrow stem cells. Using this protocol we, and others, have observed the generation of new cardiomyocytes and endothelial cells in the zone of ischemic myocardium. This approach has progressed to clinical trials at several academic institutions. Although the preliminary findings from these trials do not permit unequivocal conclusions, they do suggest that safety and feasibility are not significant problems that would argue against extending these trials in a large, randomized, double-blinded study. When considerations such as these are addressed, cell therapy may become a new modality in the treatment of heart patients.

Bone marrow stem cells regenerate infarcted myocardium.

Heart disease is the leading cause of death in the United States for both men and women. Nearly 50% of all cardiovascular deaths result from coronary artery disease. Occlusion of the left coronary artery leads to ischemia, infarction, necrosis of the affected myocardial tissue followed by scar formation and loss of function. Although myocytes in the surviving myocardium undergo hypertrophy and cell division occurs in the border area of the dead tissue, myocardial infarcts do not regenerate and eventually result in the death of the individual. Numerous attempts have been made to repair damaged myocardium in animal models and in humans. Bone marrow stem cells (BMSC) retain the ability throughout adult life to self-renew and differentiate into cells of all blood lineages. These adult BMSC have recently been shown to have the capacity to differentiate into multiple specific cell types in tissues other than bone marrow. Our research is focused on the capacity of BMSC to form new cardiac myocytes and coronary vessels following an induced myocardial infarct in adult mice. In this paper we will review the data we have previously published from studies on the regenerative capacity of BMSC in acute ischemic myocardial injury. In one experiment donor BMSC were injected directly into the healthy myocardium adjacent to the injured area of the left ventricle. In the second experiment, mice were treated with cytokines to mobilize their BMSC into the circulation on the theory that the stem cells would traffic to the myocardial infarct. In both experimental protocols, the BMSC gave rise to new cardiac myocytes and coronary blood vessels. This BMSC-derived myocardial regeneration resulted in improved cardiac function and survival.

Stem cells for myocardial regeneration.

Stem cells are being investigated for their potential use in regenerative medicine. A series of remarkable studies suggested that adult stem cells undergo novel patterns of development by a process referred to as transdifferentiation or plasticity. These observations fueled an exciting period of discovery and high expectations followed by controversy that emerged from data suggesting cell-cell fusion as an alternate interpretation for transdifferentiation. However, data supporting stem cell plasticity are extensive and cannot be easily dismissed. Myocardial regeneration is perhaps the most widely studied and debated example of stem cell plasticity. Early reports from animal and clinical investigations disagree on the extent of myocardial renewal in adults, but evidence indicates that cardiomyocytes are generated in what was previously considered a postmitotic organ. On the basis of postmortem microscopic analysis, it is proposed that renewal is achieved by stem cells that infiltrate normal and infarcted myocardium. To further understand the role of stem cells in regeneration, it is incumbent on us to develop instrumentation and technologies to monitor myocardial repair over time in large animal models. This may be achieved by tracking labeled stem cells as they migrate into myocardial infarctions. In addition, we must begin to identify the environmental cues that are needed for stem cell trafficking and we must define the genetic and cellular mechanisms that initiate transdifferentiation. Only then will we be able to regulate this process and begin to realize the full potential of stem cells in regenerative medicine.

Stem cell repair in ischemic heart disease: an experimental model.

Bone marrow stem cells (BMSC) from adult mice are now believed to generate non-hematopoietic cell types. This newly defined property is referred to as stem cell plasticity. We tested the potential of lineage negative c-kit positive (Lin- c-kit+), GFP+ BMSC to differentiate into cardiac myocytes in myocardial infarcts produced by ligation of the left coronary artery. At 9 days post-transplant the hearts showed a band of developing GFP+ myocytes within the damaged myocardium. These GFP+ myocytes were positive for cardiac specific myosin and early expressed transcription factors. Endothelial cells and smooth muscle cells also developed from the donor bone marrow cells. Left ventricular end diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP) were improved. Lin-c- kit- cells did not regenerate myocardium. We next tested the ability of cytokine-mobilized BMSC to regenerate myocardium. Nuclei in regenerating cardiomyocytes were positive for Csx/Nkx 2.5, GATA-4 and MEF2. Cytoplasmic proteins included desmin, nestin and connexin 43. Regenerating arterioles consisted of endothelial cells and smooth muscle cells positive for Ki67, and flkl. These regenerating vessels contained circulating TER119 positive red blood cells. Repair of infarcted myocardium resulted in improved heart function and survival. At day 27 after cytokine treatment and surgery, 11 of 15 mice survived compared with 9 of 52 non-treated mice. Left ventricular ejection fraction in infarcted hearts in cytokine-treated mice was 48%, 62% and 114% higher than the ejection fraction in non-treated mice at 9, 16 and 26 days following coronary artery occlusion. These findings demonstrate that circulating autologous stem cells traffic to the ischemic, infarcted myocardium and undergo differentiation into cardiomyocytes and vascular structures. We conclude that adult BMSC have the potential for repair in acute, ischemic heart disease.

Reverse Transcriptase-PCR Analysis of Gene Expression in Hematopoietic Stem Cells.

The events that determine whether hematopoietic stem cells (HSC) divide in the course of self-renewal or differentiate and become committed progenitor cells are regulated by specific gene expression. Although little is known of the molecular controls for these diverse events, the activation of a single gene may determine which developmental event will occur in an individual HSC. New and more precise information on the controls for gene expression in HSC may provide relevant clues to the regulation of hematopoiesis. Yet investigations of gene expression in HSC have been difficult to perform, primarily because it has been difficult to purify the large numbers of HSC needed to obtain sufficient RNA for Northern analysis or RNase protection assays. These rare cells occur in a ratio of approx 1:10,000 to 1:100,000 bone-marrow cells. Their enrichment is accomplished by coupling several procedures that include the use of lineage-specific monoclonal antibodies (MAbs) and immunomagnetic bead depletion of unfractionated bone marrow followed by fluorescence-activated cell sorting (FACS). The HSC in lineage-negative cell populations from mouse bone marrow are then sorted for HSC using MAbs specific for surface markers such as Sca-1 or c-kit (Fig. 1). Human HSC are lineage negative, CD34-positive and CD38-negative. Fig. 1. This scheme shows the method we use for the purification of mouse and human HSC from elutriated bone marrow. The cells in the elutriated cell fractions (FR = flow rate in mL/min) are labeled with MAbs specific for each hematopoietic lineage. These cell populations are then depleted of lineage-positive cells using immunomagnetic beads. Finally, the lineage-negative cells from mouse bone marrow are incubated with anti c-kit MAb and the lineage-negative cells from human bone marrow are incubated with anti-CD34 and anti-CD38 MAb. With a starting population of 2-4 × 10e8 unfractionated mouse bone-marrow cells, a typical isolation procedure yields approx 2 × 10e4 FR25 lin(-) c-kit(HI) cells. This is the most highly enriched HSC population we have been able to obtain to date. As few as 50- 100 of these cells can completely repopulate the hematopoietic tissue of an adult W/W(v) mouse. However, 2 × 10e4 cells from this highly enriched HSC population do not provide sufficient mRNA for Northern blots or RNase protection assays, but do provide sufficient mRNA for the RT-PCR assay.