Peroxisome proliferator-activated receptor-g agonist treatment increases septation and angiogenesis and decreases airway hyperresponsiveness in a model of experimental neonatal chronic lung disease.

Chronic lung disease (CLD) affects premature newborns requiring supplemental oxygen and results in impaired lung development and subsequent airway hyperreactivity. We hypothesized that the maintenance of peroxisome proliferator-activated receptor gamma (PPARgamma) signaling is important for normal lung morphogenesis and treatment with PPARgamma agonists could protect against CLD and airway hyperreactivity (AHR) following chronic hyperoxic exposure. This was tested in an established hyperoxic murine model of experimental CLD. Newborn mice and mothers were exposed to room air (RA) or moderate hyperoxia (70% oxygen) for 10 days and fed a standard diet or chow impregnated with the PPARgamma agonist rosiglitazone (ROSI) for the duration of study. Following hyperoxic exposure (HE) animals were returned to RA until postnatal day (P) 13 or P41. The accumulation of ROSI in neonatal and adult tissue was confirmed by mass spectrometry. Analyses of body weight and lung histology were performed on P13 and P41 to localize and quantitate PPARgamma expression, determine alveolar and microvessel density, proliferation and alpha-smooth muscle actin (alpha-SMA) levels as a measure of myofibroblast differentiation. Microarray analyses were conducted on P13 to examine transcriptional changes in whole lung. Pulmonary function and airway responsiveness were analyzed at P55. ROSI treatment during HE preserved septation and vascular density. Key array results revealed ontogeny groups differentially affected by hyperoxia including cell cycle, angiogenesis, matrix, and muscle differentiation/contraction. These results were further confirmed by histological evaluation of myofibroblast and collagen accumulation. Late AHR to methacholine was present in mice following HE and attenuated with ROSI treatment. These findings suggest that rosiglitazone maintains downstream PPARgamma effects and may be beneficial in the prevention of severe CLD with AHR.

Evidence for cell fusion is absent in vascular lesions associated with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a fatal disease associated with severe remodeling of the large and small pulmonary arteries. Increased accumulation of inflammatory cells and apoptosis-resistant cells are contributing factors. Proliferative apoptosis-resistant cells expressing CD133 are increased in the circulation of PAH patients. Circulating cells can contribute to tissue repair via cell fusion and heterokaryon formation. We therefore hypothesized that in the presence of increased leukocytes and CD133-positive (CD133(pos)) cells in PAH lung tissue, cell fusion and resulting genomic instability could account for abnormal cell proliferation and the genesis of vascular lesions. We performed analyses of CD45/CD133 localization, cell fusion, and proliferation during late-stage PAH in human lung tissue from control subjects and subjects with idiopathic (IPAH) and familial (FPAH) PAH. Localization, proliferation, and quantitation of cell populations in individual patients were performed by immunolocalization. The occurrence of cellular fusion in vascular lesions was analyzed in lung tissue by fluorescence in situ hybridization. We found the accumulation of CD45(pos) leukocytic cells in the tissue parenchyma and perivascular regions in PAH patients and less frequently observed myeloid cells (CD45/CD11b). CD133(pos) cells were detected in occlusive lesions and perivascular areas in those with PAH and were more numerous in those with IPAH lesions than in FPAH lesions. Cells coexpressing CD133 and smooth muscle alpha-actin were occasionally observed in occlusive lesions and perivascular areas. Proliferating cells were more prominent in IPAH lesions and colocalized with CD45 or CD133. We found no evidence of increased ploidy to suggest cell fusion. Taken together, these data suggest that abnormal lesion formation in PAH occurs in the absence of cell fusion.

Cryptococcus neoformans pulmonary granuloma formation is associated with matrix metalloproteinase-2 expression.

The aim of this study was to investigate matrix metalloproteinase (MMP) expression during the immune response to pulmonary Cryptococcus neoformans (Cne) infection. The immune response generated in C.B-17 and C57BL/6 mice to pulmonary Cne infection has previously been characterized as type 1 and type 2, respectively, differing in the cytokines produced and leukocytes recruited during infection, influencing the extent of Cne clearance from the lung. The focus of this study was to examine changes in expression of MMP-2 and tissue inhibitor of metalloproteinase (TIMP)-2 in the lungs of Cne-infected mice during the two types (type 1 vs. type 2) of responses. C.B-17 mice that formed well-defined granulomas had elevated levels of pulmonary MMP-2 mRNA early during infection. C57BL/6 mice that had poorly defined cellular aggregates did not express detectable levels of pulmonary MMP-2 mRNA until later in the infection. Specific expression of MMP/TIMP was correlated with the type of immune response present, resolution of Cne infection and the resulting lung pathology.

Stem cell plasticity in muscle and bone marrow.

Recent discoveries have demonstrated the extraordinary plasticity of tissue-derived stem cells, raising fundamental questions about cell lineage relationships and suggesting the potential for novel cell-based therapies. We have examined this phenomenon in a potential reciprocal relationship between stem cells derived from the skeletal muscle and from the bone marrow. We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5 day in vitro culture were harvested and introduced into each of six lethally irradiated recipients together with distinguishable whole bone marrow cells. Six and twelve weeks later, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56%, indicating that the cultured muscle cells generated approximately 10- to 14-fold more hematopoietic activity than whole bone marrow. Although the identity of the muscle-derived hematopoietic stem cells is still unknown, they may be identical to muscle satellite cells, some of which lack myogenic regulators and could respond to hematopoietic signals. We have also found that stem cells in the bone marrow can contribute to cardiac muscle repair and neovascularization after ischemic injury. We transplanted highly purified bone marrow stem cells into lethally irradiated mice that subsequently were rendered ischemic by coronary artery occlusion and reperfusion. The engrafted stem cells or their progeny differentiated into cardiomyocytes and endothelial cells and contributed to the formation of functional tissue.

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells.

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Hepatocyte growth factor expression in the developing myocardium: evidence for a role in the regulation of the mesenchymal cell phenotype and urokinase expression.

During development of the heart, the endocardium of the developing cushion tissue transforms into mesenchymal cells that rapidly invade the underlying extracellular matrix. This invasive process, along with subsequent proliferation and differentiation of the mesenchyme, are essential for the normal formation of valvular and septal structures. Several factors have been identified that appear to initiate and/or control this transformation process, including the growth factor TGF-beta. In these studies we have investigated whether hepatocyte growth factor/scatter factor (HGF/SF) may also be involved in some aspects of this transformation process. Using an immunohistochemical approach we have detected a spatially restricted localization of HGF/SF to the myocardial cells of the cushion tissue. HGF was detected in extracts of the developing heart, and the presence of the active form correlated with the onset of the transformation process and the elevation of urokinase activity. The endocardial-derived mesenchymal cells of the cushion tissue were found to express the c-met HGF receptor. Isolated endocardial cells responded to the addition of HGF with increases in motility, proliferation, and urokinase production. The results from these studies suggest that HGF may function as a myocardial-derived mediator of the epithelial-mesenchymal transformation by inducing and/or maintaining the mesenchymal cell phenotype. The increase in urokinase expression by the cushion tissue cells, in response to HGF, may facilitate the invasive abilities of these cells and also provide a means of maintaining high levels of active HGF required for the stimulation of cell proliferation and migration.

Regulation of urokinase expression in the developing avian heart: a role for the Ets-2 transcription factor.

During heart development, cells of the endocardial cushions undergo an epithelial-mesenchymal transformation and migrate into the surrounding extracellular matrix. This event is required for the normal formation of valves and chamber septation. Coincident with this phenotypic change is the expression of the serine protease urokinase by the mesenchymal cells. This protease plays an important role in remodeling of the matrix, promotion of cell migration by regulating cell-matrix interactions, and the activation of growth factors. To understand the mechanisms underlying the expression of urokinase during heart development, studies were designed to analyze the role of the Ets transcription factors in the regulation of the avian urokinase gene promoter. Deletion or mutagenesis of the Ets consensus sites significantly decreased the activity of the promoter in isolated cushion tissue cells. Proteins were identified by electrophoretic mobility shift analysis and UV-crosslinking which bound to a specific region of the promoter shown to be required for full transcriptional activity. Analyses based upon protein molecular weight and interaction with specific antibodies suggest a role for the Ets-2 protein in promoter binding and activity. The expression of Ets-2 in the cushion tissue cells was confirmed by RT-PCR analysis and in situ hybridization. The mRNA levels and the DNA binding activity of the Ets-2 protein were found to change during development paralleling the increase in urokinase activity. Overexpression of the full-length Ets-2 protein or a dominant-negative form of the protein altered the activity of the promoter and significantly affected the production of urokinase in these cells. The results from these studies suggest an important role for the Ets-2 protein in heart development and may contribute to a better understanding of the inductive factors present in the heart which facilitate the normal morphogenesis of this organ.