Hypoxic induction of interleukin-8 gene expression in human endothelial cells.

Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.

Hypoxia-mediated induction of endothelial cell interleukin-1 alpha. An autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface.

Tissue injury that accompanies hypoxemia/reoxygenation shares features with the host response in inflammation, suggesting that cytokines, such as IL-1, may act as mediators in this setting. Human endothelial cells (ECs) subjected to hypoxia (PO2 approximately 12-14 Torr) elaborated IL-1 activity into conditioned media in a time-dependent manner; this activity was completely neutralized by an antibody to IL-1 alpha. Production of IL-1 activity by hypoxic ECs was associated with an increase in the level of mRNA for IL-1 alpha, and was followed by induction of endothelial-leukocyte adhesion molecule-1 and enhanced expression of intercellular adhesion molecule-1 (ICAM-1) during reoxygenation. During reoxygenation there was a three- to five-fold increased adherence of leukocytes, partly blocked by antibodies to endothelial-leukocyte adhesion molecule-1 and ICAM-1. Suppressing endothelial-derived IL-1, using either antibodies to IL-1 alpha, specific antisense oligonucleotides or the IL-1 receptor antagonist, decreased leukocyte adherence to reoxygenated ECs, emphasizing the integral role of IL-1 in the adherence phenomenon. Mice subjected to hypoxia (PO2 approximately 30-40 Torr) displayed increased plasma levels of IL-1 alpha, induction of IL-1 alpha mRNA in the lung, and enhanced expression of ICAM-1 in pulmonary tissue compared with normoxic controls. These data suggest that hypoxia is a stimulus which induces EC synthesis and release of IL-1 alpha, resulting in an autocrine enhancement in the expression of adhesion molecules.

Adhesion molecules (E-selectin and ICAM-1) in pulmonary allograft rejection.

Vascular endothelial cells act as antigen-presenting cells in the lung allograft and stimulate alloreactive host lymphocytes. Activated lymphocytes and cytokines can induce expression of leukocyte-endothelial adhesion molecules that facilitate invasion of the allograft by circulating leukocytes. To define the role of endothelial HLA class II antigen and adhesion molecule expression in lung allograft rejection, we prospectively analyzed endothelial expression of HLA class II, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) antigens in 52 transbronchial biopsy specimens from 24 lung allograft recipients as compared to normal control subjects. Thirty-one of 52 specimens showed histologic rejection and 8 of 24 patients developed histologic obliterative bronchiolitis (OB) by the end of the study period. Increased expression of HLA class II antigen was seen in 32 of 52 (62%) lung allograft specimens, but increased expression did not correlate with acute rejection or OB. In contrast, E-selectin expression was seen in 30 of 52 (58%) biopsy specimens and was associated with acute rejection (p < 0.005) and with the development of OB (p < 0.05). Increased expression of ICAM-1 was seen in only 18 of 52 (35%) biopsy specimens and did not correlate with acute rejection or OB. These data suggest that E-selectin expression may be a tissue marker of acute and chronic lung rejection possibly by promoting leukocyte adhesion to the allograft endothelium. The high levels of endothelial HLA class II expression may reflect long-term antigenic stimulation of the allograft even in the absence of rejection.

Electrophysiologic effects of a novel selective adenosine A1 agonist (CVT-510) on atrioventricular nodal conduction in humans.

BACKGROUND: CVT-510, N-(3(R)-tetrahydrofuranyl)-6-aminopurine riboside, is a selective A(1)-adenosine receptor agonist with potential potent antiarrhythmic effects in tachycardias involving the atrioventricular (AV) node. This study, the first in humans, was designed to determine the effects of CVT-510 on AV nodal conduction and hemodynamics. METHODS AND RESULTS: Patients in sinus rhythm with normal AV nodal function at electrophysiologic study (n = 32) received a single intravenous bolus of CVT-510. AH and HV intervals were measured during sinus rhythm and during atrial pacing at 1, 5, 10, 15, 20, 30, 45, and 60 minutes after the bolus. Increasing doses of CVT-510 (0.3 to 10 microg/kg) caused a dose-dependent increase in the AH interval. At 1 minute, a dose of 10 microg/kg increased the AH interval during sinus rhythm from 93 +/- 23 msec to 114 +/- 37 msec, p = 0.01 and from 114 +/- 31 msec to 146 +/- 44 msec during atrial pacing at 600 msec, p = 0.003). The AH interval returned to baseline by 20 minutes. CVT-510 at doses of 0.3 to 10 microg/kg had no effect on sinus rate, HV interval, or systemic blood pressure, and was not associated with serious adverse effects. At doses of 15 and 30 microg/kg, CVT-510 produced transient second/third degree AV heart block in all four patients treated. One of these patients also had a prolonged sedative effect that was reversed with aminophylline. CONCLUSIONS: CVT-510 promptly prolongs AV nodal conduction and does not affect sinus rate or blood pressure. Selective stimulation of the A(1)-adenosine receptor by CVT-510 may be useful for immediate control of heart rate in atrial fibrillation/flutter and to convert paroxysmal supraventricular tachycardia to sinus rhythm, while avoiding vasodilatation mediated by the A(2)-adenosine receptor, as well as the vasodepressor and negative inotropic effects associated with beta-adrenergic receptor blockade and/or calcium channel blockers.

Modulation of endothelial function by hypoxia: perturbation of barrier and anticoagulant function, and induction of a novel factor X activator.

Exposure of the vessel wall to hypoxemia is a central feature of ischemic cardiovascular disease. This led us to examine the perturbation of endothelial cell properties under hypoxia. An atmosphere of pO2 of 12 mmHg is not lethal to the endothelial cells for up to five days, but barrier function was impaired. Increased passage of macromolecule tracers were observed in time- and dose-dependent manner and electron microscopy demonstrated small gaps (0.5-1.0 micron) between cells. Expression of the anticoagulant cofactor thrombomodulin was also perturbed: thrombomodulin activity and antigen decreased in parallel. Northern blots showed almost complete suppression of thrombomodulin in hypoxic culture. Furthermore, synthesis of other proteins, such as fibronectin, was slightly enhanced under hypoxia. In addition to the suppression of these anticoagulant cofactor, hypoxic endothelial cell displayed a noval procoagulant activity distinct from tissue factor. Further study revealed that hypoxic endothelial cultures directly activated Factor X, as assessed by functional assays and SDS-PAGE. In addition to this no activation of Factor IX or prothrombin was observed. The hypoxia-induced Factor X activator was membrane-associated, required calcium to form Factor Xa, was inhibited by HgCl2 but not by PMSF, and had Km approximately 25 micrograms/ml. Co-incubation of hypoxic cultures with cycloheximide prevented the expression of this activity, suggesting that protein synthesis is required for its expression. These functional perturbations of endothelial cells were reversible following reoxygenation. These data indicate that hypoxia imposes a selective perturbation on endothelial cell function, suggesting the possible contribution of hypoxemia to vascular dysfunction in ischemia.

Hypoxia induces endothelial cell synthesis of membrane-associated proteins.

Hypoxemia is associated with a prothrombotic tendency. In this study we report the purification and partial characterization of an activator of a central coagulation component, factor X, induced in endothelium by exposure to hypoxia (hypoxia-induced factor X activator or Xact). Expression of Xact occurred in a reversible manner when endothelial cell cultures were exposed to hypoxia or sodium azide but not in response to a variety of other alterations in the cellular milieu, such as heat shock or glucose deprivation. The activity of Xact, which was not detected in normoxic endothelial cells, was maximal under acidic conditions, pH 6.0-6.8, which often coexist with hypoxia in an ischemic milieu. By sequential isoelectric focusing and preparative SDS/PAGE of endothelial membrane-rich fractions, Xact was purified approximately 19,000-fold and found to be a single-chain, approximately 100-kDa polypeptide with pI approximately 5.0. Activation of factor X by purified Xact was not affected by blocking antibodies to other coagulation proteins or by phenylmethylsulfonyl fluoride or leupeptin but was prevented by mercury chloride or iodoacetamide. In addition to the induction of Xact, two-dimensional gel analysis of membrane fractions from metabolically labeled hypoxic endothelial cultures revealed two groups of approximately 10 additional spots: (i) a group for which expression was maximal after 24 hr and (ii) a group for which expression continued to increase up to 48 hr. The pattern of hypoxia-mediated modulation of protein expression was distinct from that seen with other cellular stimuli but could be duplicated, in part, by sodium azide. These results indicate that hypoxia elicits a specific biosynthetic response, including the expression of endothelial cell-surface molecules that can alter cellular function and may potentially serve as markers of hypoxemic vessel-wall injury.

Opportunistic bronchopulmonary infections after lung transplantation: clinical and radiographic findings.

PURPOSE: To assess clinical and radiographic findings in opportunistic bronchopulmonary infections after lung transplantation. MATERIALS AND METHODS: Forty-five episodes of opportunistic bronchopulmonary infection occurred in 27 (35%) of 77 lung transplant recipients during a 4-year period. Causative organisms, radiographic patterns, and mortality were reviewed. RESULTS: Cytomegalovirus (CMV) was the most common pathogen (25 episodes), followed by Aspergillus species (seven episodes), Pneumocystis carinii (six episodes), herpes simplex virus (four episodes), Mycobacterium avium complex (two episodes), and M tuberculosis (one episode). Eighteen of the 25 episodes (72%) of CMV pneumonitis occurred in the first 4 months after transplantation; 24 (96%) occurred within the 1st year. Radiographic patterns of symptomatic CMV pneumonitis were diffuse haziness (60%), focal haziness (33%), and focal consolidation (7%). Aspergillus species locally invaded a necrotic bronchial anastomosis in three patients, each within 4 months of transplantation. P carinii was seen as focal haziness and caused no symptoms. Radiographic findings, when present, were seen almost exclusively in the transplanted lung. Despite three deaths attributable to opportunistic bronchopulmonary infection, the difference between the survival rates of patients with and those of patients without bronchopulmonary infection was not statistically significant (82% and 81%, respectively, 1 year after transplantation). CONCLUSION: Opportunistic bronchopulmonary infections are common after lung transplantation. The most common pathogen is CMV, which causes diverse chest radiographic patterns. Opportunistic bronchopulmonary infections do not adversely affect overall mortality.

The effect of hypoxia on capillary endothelial cell function: modulation of barrier and coagulant function.

As the cells forming the luminal vascular surface, endothelium is exposed to alterations in the vascular microenvironment, such as hypoxaemia. In this report we demonstrate that hypoxia, with pO2 as low as 12-14 mmHg, was not toxic to endothelium, but reversibly modulated central cellular functions essential for maintenance of homeostasis: permeability of monolayers to solutes increased in a dose-dependent manner, and cell surface coagulant properties were shifted to promote activation of coagulation. The anticoagulant cofactor thrombomodulin was suppressed and an apparently novel activator of factor X, distinct from the classical extrinsic and intrinsic systems, was induced. The hypoxia-induced factor X activator was cell surface-associated, had properties of a cysteine protease, had Mr corresponding to approximately 100 kDa, based on sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE), and isoelectric point (pI) approximately 5.0. These findings indicate that hypoxia dynamically modulates endothelial function providing insights into the contribution of microvascular endothelial dysfunction in the pathogenesis of vascular lesions.

Macrovascular and microvascular endothelium during long-term hypoxia: alterations in cell growth, monolayer permeability, and cell surface coagulant properties.

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a approximately 3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro- and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.