Faculty Bibliography

BACKGROUND AND PURPOSE: Neutrophil (PMN) recruitment mediated by increased expression of intercellular adhesion molecule-1 expression (ICAM-1, CD54) in the cerebral microvasculature contributes to the pathogenesis of tissue injury in stroke. However, studies using blocking antibodies against the common beta2-integrin subunit on the PMN, the counterligand for ICAM-1 (CD18), have demonstrated equivocal efficacy. The current study tested the hypothesis that mice deficient in CD18 would be protected in the setting of reperfused but not nonreperfused stroke. METHODS: Two groups of mice were studied, those whose PMNs could express CD18 (CD18 +/+) and those mice hypomorphic for the CD-18 gene (CD18 -/-). PMNs obtained from CD18 -/- or CD18 +/+ mice were fluorescently labeled and tested for binding to murine brain endothelial monolayers. Using a murine model of focal cerebral ischemia in which an occluding suture placed in the middle cerebral artery (MCA) is removed after 45 minutes (transient ischemia, reperfused stroke) or left in place (permanent ischemia, nonreperfused stroke), cerebral infarct volumes (% ipsilateral hemisphere by TTC staining), cerebral blood flow (CBF, % contralateral hemisphere by laser-Doppler flowmetry), and survival (%) were examined 24 hours after the initial ischemic event. Adoptive transfer studies used 111In-labeled PMNs (from either CD18 +/+ or CD18 -/- mice) to examine the relative accumulation of PMNs in the ischemic region. RESULTS: PMNs obtained from CD18 -/- mice exhibit reduced adhesivity (compared with CD18 +/+ PMNs) for both quiescent and cytokine-activated endothelial monolayers. CD18 -/- mice (n=14) subjected to transient focal cerebral ischemia demonstrated a 53% decrease in infarct volumes versus CD18 +/+ mice (n=26, P<0.05), improved penumbral CBF at 24 hours (1.8-fold, P=0.02), and a 3.7-fold decrease in mortality (P=0.02). However, when CD18 -/- mice (n=12) were subjected to permanent focal cerebral ischemia, no differences were noted in infarct volume, mortality, or CBF versus similarly treated CD18 +/+ mice (n=10). There was a greater accumulation of CD18 +/+ PMNs in the ischemic zone of CD18 +/+ animals than CD18 -/- animals subjected to reperfused stroke (82% increase, P=0.02), although there was no difference between groups when subjected to permanent MCA occlusion. CONCLUSIONS: Deficiency for the CD18 gene confers cerebral protection in a murine model of reperfused stroke, but this benefit does not extend to CD18-deficient animals subjected to permanent MCA occlusion. These data suggest that anti-PMN strategies should be targeted to reperfused stroke and may perhaps be used in conjunction with thrombolytic therapy that establishes reperfusion

Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 approximately 13 torr) had increased levels of tissue factor transcripts (approximately 18-fold) and an increased rate of transcription (approximately 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; -111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role

The lung is the major site expressing plasma phospholipid transfer protein (PLTP) mRNA in humans and mice, suggesting that this protein might have an important role in maintaining normal function of this organ. In the lung of human collagenase transgenic mice, an emphysematous animal model, PLTP mRNA was 3-fold higher than in control mice. However, the mRNA in other tissues was not changed. To further assess the expression and function of PLTP, we measured PLTP mRNA level in lung tissue of two emphysematous patients and found that the mRNA was 4-fold higher than in control subjects. In situ hybridization on mouse lung suggested positive staining in alveolar type II epithelial cells. In addition, immortalized rat alveolar pre-type II epithelial cells and freshly isolated mature rat alveolar type II epithelial cells both highly expressed PLTP mRNA, and the former cells actively secreted PLTP activity into the medium. To examine the possible mechanisms leading to high levels of PLTP expression in vivo, we exposed the pre-type II cells to hypoxia and demonstrated induction of PLTP mRNA and a coordinate increase in secreted PLTP activity. Thus, the PLTP gene is highly expressed in alveolar type II epithelial cells and is induced during hypoxia and in emphysema. These observations suggest that a hypoxic stimulus occurring in emphysema may be a novel mechanism that contributes to enhanced expression of PLTP

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Abeta) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Abeta, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappaB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Abeta deposits, and in cerebrospinal fluid from AD patients there was approximately 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Abeta-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Abeta, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Abeta on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD

Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis

Activation of transcription at the nuclear factor interleukin 6 (NF-IL-6) DNA binding motif modulates expression of multiple genes important in host adaptive and developmental mechanisms. Studies showing that hypoxia-induced transcription of IL-6 in cultured endothelial cells was due to transcriptional activation by the NF-IL-6 motif in the promoter (Yan, S.-F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L., and Stern, D. (1995) J. Biol. Chem. 270, 11463-11471) led us to prepare transgenic mice using 115- or 14-base pair regions of the promoter encompassing the NF-IL-6 site ligated to the lacZ reporter gene and the basal thymidine kinase promoter. On exposure to hypoxia or induction of ischemia, mice bearing either of the constructs showed prominent expression of the transgene in lung and cardiac vasculature and in the kidney but not in the liver (parenchyma or vasculature). In contrast, transgenic mice bearing a mutationally inactivated NF-IL-6 site showed no increase in transgene expression in hypoxia. Gel retardation assays revealed time-dependent, hypoxia-enhanced nuclear binding activity for the NF-IL-6 site in nuclear extracts of the heart, lung, and kidney but not in the liver; the hypoxia-enhanced band disappeared on addition of antibody to C/EBPbeta-NF-IL-6. Consistent with the specificity of hypoxia-mediated activation of C/EBPbeta-NF-IL-6, gel retardation assays showed no change in the intensity of the hypoxia-enhanced gel shift band in the presence of excess unlabeled oligonucleotide probes or antibodies related to other transcription factors, including NFkappaB, AP1, cAMP response element-binding protein, SP1, and hypoxia-inducible factor 1. These data indicate that the transcription factor NF-IL-6 is sensitive to environmental oxygen deprivation, and the tissue-specific pattern of gene expression suggests that local mechanisms have an important regulatory effect

The endothelial cell response to hypoxia involves a range of adaptive mechanisms that reflect an active response of the cell's biosynthetic and metabolic apparatus. Hypoxia-mediated suppression of endothelial barrier function, resulting in increased vascular leakage, is likely to contribute to pulmonary and cerebral edema associated with high altitude and is closely associated with a fall in intracellular cyclic AMP levels. Buttressing of this second messenger pathway in the endothelium using membrane permeant cyclic AMP analogs prevents increased vascular leakage due to hypoxia. Application of this principle to organ preservation has shown that supplementation with cyclic AMP analogs or inhibition of endogenous cAMP metabolism enables extension of the time a harvested organ can remain extracorporeally, after which transplantation is successful. The underlying mechanism through which cyclic AMP exerts its effects appears to be maintenance of vascular homeostasis in the graft. A distinct adaptive mechanism triggered in the endothelium by hypoxia is expression of the cytokine interleukin-6 (IL-6) by a novel mechanism involving transcription driven by the nuclear factor IL-6 (NF-IL-6) DNA binding site in the promoter. IL-6 may exert protective effects on vascular function, thereby limiting vascular injury by a different mechanism than those recruited by elevated cAMP levels. These studies provide insights into tow independent mechanisms through which endothelium responds to oxygen deprivation, and suggest possible new approaches to attentuate vascular injury associated with ischemia