Ligand-dependent regulation of vascular endothelial growth factor and erythropoietin expression by a plasmid-based autoinducible GeneSwitch system.

We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate the expression of genes for two therapeutic proteins: vascular endothelial growth factor (VEGF) and erythropoietin. The GeneSwitch system consisted of two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene. When the constitutive CMV promoter of the GeneSwitch plasmid was replaced by an autoinducible promoter consisting of four copies of GAL4 DNA binding sites linked to a minimal thymidine kinase promoter, the tightness of transgene regulation was improved by an order of magnitude. Quantitative RT-PCR analysis of GeneSwitch mRNA confirmed that the autoinducible promoter was responsive to mifepristone. We demonstrated the ability of the improved GeneSwitch system to regulate the expression of VEGF or erythropoietin in a biologically relevant manner after delivery of plasmids to the hind-limb muscle of adult mice. This ability of the autoinducible GeneSwitch system to regulate the expression of therapeutic proteins in mice indicates its potential for use in human gene therapy applications.

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.

Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion.

BACKGROUND: Myocardial infarction is associated with an intense inflammatory reaction leading to healing and scar formation. Because mast cells are a significant source of fibrogenic factors, we investigated mast cell accumulation and regulation of stem cell factor (SCF), a potent growth and tactic factor for mast cells, in the healing myocardium. METHODS AND RESULTS: Using a canine model of myocardial ischemia and reperfusion, we demonstrated a striking increase of mast cell numbers during the healing phase of a myocardial infarction. Mast cell numbers started increasing after 72 hours of reperfusion, showing maximum accumulation in areas of collagen deposition (12.0+/-2.6-fold increase; P<0.01) and proliferating cell nuclear antigen (PCNA) expression. The majority of proliferating cells were identified as alpha-smooth muscle actin-positive myofibroblasts or factor VIII-positive endothelial cells. Mast cells did not appear to proliferate. Using a nuclease protection assay, we demonstrated induction of SCF mRNA within 72 hours of reperfusion. Immunohistochemical studies demonstrated that a subset of macrophages was the source of SCF immunoreactivity in the infarcted myocardium. SCF protein was not found in endothelial cells and myofibroblasts. Intravascular tryptase-positive, FITC-avidin-positive, CD11b-negative mast cell precursors were noted in the area of healing and in the cardiac lymph after 48 to 72 hours of reperfusion. CONCLUSIONS: Mast cells increase in number in areas of collagen deposition and PCNA expression after myocardial ischemia. The data provide evidence of mast cell precursor infiltration into the areas of cellular injury. SCF is induced in a subset of macrophages infiltrating the healing myocardium. We suggest an important role for SCF in promoting chemotaxis and growth of mast cell precursors in the healing heart.

Molecular evidence for induction of intracellular adhesion molecule-1 in the viable border zone associated with ischemia-reperfusion injury of the dog heart.

BACKGROUND: Acute inflammation may play a role in injury during reperfusion following myocardial ischemia. Studies in vitro suggest that intracellular adhesion molecule-1 (ICAM-1) mediates neutrophil adherence to cardiac myocytes and neutrophil-mediated injury. We have shown cytokine activity in postischemic cardiac lymph sufficient to maximally express ICAM-1 on myocytes and that ICAM-1 mRNA is found in the previously ischemic myocardium early in reperfusion. METHODS AND RESULTS: In the present study, we used in situ hybridization techniques to detect ICAM-1 mRNA and examine the cells of origin, relation to cell injury, and relation to inflammatory infiltration after 1 hour of ischemia and varying times of reperfusion. By 1 hour of reperfusion, ICAM-1 mRNA was detected in much of the ischemic myocardium, except in areas of contraction band necrosis. At 2 and 3 hours, a clear demarcation of necrotic areas surrounding ischemic areas of viable myocardium with ICAM-1 mRNA staining was present, and ICAM-1 mRNA staining increased with time. Nonischemic areas had no visible ICAM-1 mRNA staining in the first 3 hours. By 24 hours of reperfusion, ICAM-1 mRNA was present in both control and ischemic segments (excluding the necrotic areas) compatible with a generalized circulation of cytokines persistent at 24 hours. In the absence of reperfusion, ICAM-1 mRNA staining was not seen in the first 3 hours and was markedly reduced at 24 hours. The interface of viable and necrotic cells also contained the most extensive inflammatory infiltration. CONCLUSIONS: Evidence is presented that induction of ICAM-1 mRNA has highly specific localization to ischemic but viable myocardium. Induction of ICAM-1 mRNA transcription in early reperfusion may render the viable "border zone" susceptible to neutrophil-induced injury.

Molecular evidence for a border zone vulnerable to inflammatory reperfusion injury.

Acute inflammation has been suggested as a potential mechanism for some of the injury associated with reperfusion of the ischemic myocardium. This hypothesis implies that viable myocardial cells adjacent to the lethally injured cells are vulnerable to injury induced by the neutrophil influx observed to attend reperfusion. In our previous work, we demonstrated that the presence of ICAM-1 on the surface of cardiac myocytes is required for neutrophils to directly damage them; blocking monoclonal antibodies to either ICAM-1 on cardiac myocytes or Mac-1 on activated neutrophils completely precluded neutrophil-induced myocyte injury. We also demonstrated that postischemic cardiac lymph (cardiac extracellular fluid) contained leukotactic factors (primarily C5a) and cytokines present in concentrations sufficient to maximally induce Mac-1 on the surface of neutrophils and ICAM-1 on the surface of isolated dog cardiac myocytes. The present study sought to further these observations by examining the site of potential ICAM-1 induction as a function of time of reperfusion, degree of ischemia, and viability of myocardial cells. Our evidence suggests that ICAM-1 mRNA is induced very early after reperfusion only in the previously ischemic myocardium and is not seen in the nonischemic myocardium during the early hours of reperfusion. Moreover, ICAM-1 mRNA induction is seen most intensely in the ischemic area directly bordering the necrotic area (which, after 1-hr reperfusion, does not contain any ICAM-1 mRNA) and immediately abutting the site of maximal influx of neutrophils. Thus, the induction of ICAM-1 and the influx of neutrophils (presumably activated by the chemotactic factors that guided their migration) exists on the border between viable and necrotic cells. This provides the first direct molecular evidence for a jeopardized border zone on the edge of myocardial infarction during reperfusion. As previously demonstrated, this reaction is wholly dependent upon tissue injury of the ischemic myocardium and therefore represents an example of a mechanism of injury extension induced as a reaction to a primary injury. The degree of specificity of this reaction demonstrated by the subendocardial sparing directly adjacent to ischemic cells suggests finely modulated mechanisms by which this process is controlled.