Reactive oxygen species and p38 mitogen-activated protein kinase mediate tumor necrosis factor α-converting enzyme (TACE/ADAM-17) activation in primary human monocytes.

Tumor necrosis factor α-converting enzyme (TACE) is responsible for the shedding of cell surface TNF. Studies suggest that reactive oxygen species (ROS) mediate up-regulation of TACE activity by direct oxidization or modification of the protein. However, these investigations have been largely based upon nonphysiological stimulation of promonocytic cell lines which may respond and process TACE differently from primary cells. Furthermore, investigators have relied upon TACE substrate shedding as a surrogate for activity quantification. We addressed these concerns, employing a direct, cell-based fluorometric assay to investigate the regulation of TACE catalytic activity on freshly isolated primary human monocytes during LPS stimulation. We hypothesized that ROS mediate up-regulation of TACE activity indirectly, by activation of intracellular signaling pathways. LPS up-regulated TACE activity rapidly (within 30 min) without changing cell surface TACE expression. Scavenging of ROS or inhibiting their production by flavoprotein oxidoreductases significantly attenuated LPS-induced TACE activity up-regulation. Exogenous ROS (H(2)O(2)) also up-regulated TACE activity with similar kinetics and magnitude as LPS. H(2)O(2)- and LPS-induced TACE activity up-regulation were effectively abolished by a variety of selective p38 MAPK inhibitors. Activation of p38 was redox-sensitive as H(2)O(2) caused p38 phosphorylation, and ROS scavenging significantly reduced LPS-induced phospho-p38 expression. Inhibition of the p38 substrate, MAPK-activated protein kinase 2, completely attenuated TACE activity up-regulation, whereas inhibition of ERK had little effect. Lastly, inhibition of cell surface oxidoreductases prevented TACE activity up-regulation distal to p38 activation. In conclusion, our data indicate that in primary human monocytes, ROS mediate LPS-induced up-regulation of TACE activity indirectly through activation of the p38 signaling pathway.

Mobilization and margination of bone marrow Gr-1high monocytes during subclinical endotoxemia predisposes the lungs toward acute injury.

The specialized role of mouse Gr-1(high) monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during subclinical endotoxemia in mice and investigated whether marginated monocytes contribute to lung injury in response to further septic stimuli. Subclinical low-dose i.v. LPS induced a rapid (within 2 h), large-scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these premarginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the preadministration of low-dose LPS. The LPS-zymosan-induced permeability increases were effectively abrogated by pretreatment (30 min before zymosan challenge) with the platelet-activating factor antagonist WEB 2086 in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of platelet-activating factor/ceramide-mediated pathways in this model. Depletion of monocytes (at 18 h after clodronate-liposome treatment) significantly attenuated the LPS-zymosan-induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48 h after clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during subclinical endotoxemia primes the lungs toward further septic stimuli and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.

Functional and morphological maturation of implanted neonatal cardiomyocytes as a comparator for cell therapy.

Knowledge of the rate of development of immature cardiomyocytes after implantation into a host heart is important for studies using cell therapy. To assess this functionally, we have implanted rat neonatal cardiomyocytes (NCMs) in normal and infarcted rat heart and re-isolated them for functional assessment. Maturation of implanted bone marrow stromal cells (BMSCs) was compared under similar conditions. NCMs from green fluorescent protein (GFP) transgenic rats were implanted into adult normal or infarcted rat hearts and re-isolated after 1, 2, or 4 weeks by standard enzymatic digestion. BMSCs labeled with DiI and iron oxide were implanted into rats with myocardial infarction and cells re-isolated 1, 2, 5, 6, and 16 weeks later. GFP-labeled myocytes approaching the adult morphology were detected 2 weeks after implantation of NCMs, but were significantly shorter than adult host myocytes and had reduced contractility. By 4 weeks after implantation, re-isolated GFP-labeled myocytes were close to the adult phenotype in contractile characteristics, although still significantly shorter. Infarction of the host did not alter the rate of maturation of implanted cells. After implantation of BMSCs, small numbers of functional DiI-labeled myocytes were re-isolated from 4/11 animals but were more mature than expected from the NCM studies. This adds evidence that BMSC-derived cardiomyocytes were not a result of transdifferentiation. The maturation rate of implanted NCMs represents a benchmark against which to evaluate the likely rate of formation of fully functional cardiomyocytes from implanted cells.

The impact of proliferative potential of umbilical cord-derived endothelial progenitor cells and hypoxia on vascular tubule formation in vitro.

Revascularization of the damaged tissue is pivotal to tissue repair. Here, by bringing together two in vitro model systems, we have been able to examine (1) the ability of human umbilical vein endothelial cells (HUVEC) containing a complete hierarchy of endothelial progenitors derived from the human umbilical cord to generate vascular tubules within a human stromal niche in vitro and (2) the effects of exposure to low oxygen tensions on endothelial progenitor cell proliferation and tubule formation in vitro. Our results demonstrate that high proliferative potential endothelial colony forming cells (HPP-ECFC) from cultured HUVEC preferentially contribute to vascular tubule formation in vitro and that these progenitor cells are concentrated in the CD34(lo/-) fraction. HUVEC were initially resistant when exposed to hypoxia (1.5% O(2)) for short periods (1-2 days), but sustained chronic hypoxia (4-14 days) inhibited their ability to proliferate. This was reflected by a loss in their ability to form tubules in cocultures of human dermal fibroblasts (hDFs). In contrast, an acute exposure to low oxygen tensions (1.5% O(2) for 24 h) followed by reoxygenation did not adversely affect the capacity of these cells to both proliferate and form vascular tubules in vitro.These studies therefore provide a model system to study the influences of the microenvironmental niche and modification of this niche on vascular tubule formation in vitro from HPP-ECFC.

Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: an in vivo cine-MRI study.

Basic and clinical studies have shown that bone marrow cell therapy can improve cardiac function following infarction. In experimental animals, reported stem cell-mediated changes range from no measurable improvement to the complete restoration of function. In the clinic, however, the average improvement in left ventricular ejection fraction is around 2% to 3%. A possible explanation for the discrepancy between basic and clinical results is that few basic studies have used the magnetic resonance (MR) imaging (MRI) methods that were used in clinical trials for measuring cardiac function. Consequently, we employed cine-MR to determine the effect of bone marrow stromal cells (BMSCs) on cardiac function in rats. Cultured rat BMSCs were characterized using flow cytometry and labeled with iron oxide particles and a fluorescent marker to allow in vivo cell tracking and ex vivo cell identification, respectively. Neither label affected in vitro cell proliferation or differentiation. Rat hearts were infarcted, and BMSCs or control media were injected into the infarct periphery (n = 34) or infused systemically (n = 30). MRI was used to measure cardiac morphology and function and to determine cell distribution for 10 wk after infarction and cell therapy. In vivo MRI, histology, and cell reisolation confirmed successful BMSC delivery and retention within the myocardium throughout the experiment. However, no significant improvement in any measure of cardiac function was observed at any time. We conclude that cultured BMSCs are not the optimal cell population to treat the infarcted heart.

Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron labeling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine-magnetic resonance imaging (cine-MRI) and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterized by flow cytometry, transduced with lentiviral vectors expressing green fluorescent protein (GFP), and labeled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation, and rat hearts were imaged at 1, 4, 10, and 16 weeks postinfarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whereas the signal void volume did not decrease significantly in severely infarcted hearts. High-resolution three-dimensional magnetic resonance (MR) microscopy identified hypointense regions at the same position as in vivo. Donor cells containing iron particles and expressing GFP were identified in MR-targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labeled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron-labeled donor cells can be used for their isolation from host tissue to enable further characterization.

The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases.

4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit. While investigating the role of Src kinases in SCF signaling, we found that PP1 completely abrogated the proliferation of M07e cells in response to SCF. PP1 inhibited SCF-induced c-Kit autophosphorylation in intact cells and blocked the activation of mitogen-activated protein kinase and Akt. In vitro kinase assays using immunoprecipitated c-Kit confirmed direct inhibition by PP1. SCF-induced c-Kit phosphorylation was also inhibited by the related inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) and by STI571 but not by the Src inhibitor SU6656. PP1 inhibited the activity of mutant constitutively active forms of c-Kit (D814V and D814Y) found in mast cell disorders, and triggered apoptosis in the rat basophilic leukemia cell line RBL-2H3 that expresses mutant c-Kit. In addition, PP1 (and PP2) inhibited the in vitro kinase activity and autophosphorylation in whole cells of p210 Bcr-Abl. PP1 reduced the constitutive activation of signal transducer and activators of transcription 5 and mitogen-activated protein kinase and triggered apoptosis in FDCP1 cells expressing Bcr-Abl. These results have implications for the use of PP1 in investigating intracellular signaling and suggest that PP1 or related compounds may be useful in the treatment of malignant diseases associated with dysregulated c-Kit or Abl tyrosine kinase activity.