Alpha A. Fowler

Hypoxia Inducible Factor-1 Activation by Prolyl 4-Hydroxylase-2 Gene Silencing Attenuates Myocardial Ischemia Reperfusion Injury

Hypoxia inducible factor-1 (HIF-1) regulates changes in transcription of key genes such as inducible NO synthase (iNOS) in hypoxic/ischemic environments. In normoxia, HIF-1 activation is controlled by HIF-1&agr;-prolyl 4-hydroxylases, which target HIF-1&agr; for ubiquitination and proteasomal degradation. We hypothesized that normoxic HIF-1 preservation could attenuate cardiac ischemia/reperfusion injury via a preconditioning effect. HIF-1 preservation was achieved by using small interfering RNA (siRNA) to silence murine HIF-1&agr;-prolyl-4 hydroxylase-2 (PHD2). PHD2 siRNA reduced PHD2 mRNA expression 89±1.5% (P<0.001) in a time- and concentration-dependent manner in normoxic murine microvascular endothelial cells (EC). PHD2 silencing in normoxic EC stabilized HIF-1&agr; protein levels while significantly increasing HIF-1 transcriptional activity and iNOS mRNA expression. Wild-type mice infused with PHD2 siRNA (1.5 &mgr;g/g body weight) showed a 61±2.4% (P<0.05) reduction in cardiac PHD2 mRNA within 24 hours. In addition HIF-1&agr; protein levels and HIF-1-dependent iNOS mRNA levels were increased. PHD2 siRNA-transfected hearts from wild-type mice (n=6) subjected to 30 minutes ischemia followed by 60 minutes reperfusion exhibited reduced infarct size when compared with saline-treated controls (9.7±1.9% versus 31.6±1.8%, respectively, P<0.0001, n=6) and to control mice transfected with a nontargeting siRNA control (28.4±3.0%, P<0.0001, n=6). Hearts from iNOS knockout mice receiving PHD2 siRNA by identical injection protocol (n=6) exhibited infarct size indistinguishable from saline controls (28.7±1.3%). These results show that in vitro and in vivo, PHD2 silencing using a siRNA strategy produces transcriptionally active HIF-1. Normoxic activation of HIF-1 in hearts following in vivo PHD2 siRNA administration attenuates reperfusion injury via an iNOS-dependent pathway.

CD18 adhesion receptors, tumor necrosis factor, and neutropenia during septic lung injury☆

Sequestration of neutrophils (PMNs) in the pulmonary microvasculature and associated neutropenia are characteristic features of experimental models of septic lung injury. The etiology of altered PMN kinetics during septic lung injury is uncertain, but may be partially due to increased adhesiveness of activated PMNs to pulmonary endothelium. This study examines the relationship between the expression of PMN CD18 adhesion receptors, the evolving neutropenia, and plasma tumor necrosis factor (TNF) activity in a porcine model of septic lung injury. Acute lung injury was induced by infusion of live Pseudomonas aeruginosa (5 x 10(8) CFU/ml at 0.3 ml/20 kg/min) for 60 min (Group Ps, n = 6). Control animals (Group C, n = 3) received a 60-min infusion of sterile 0.9% saline. CD18 expression of circulating PMNs was measured by quantitative immunofluorescent flow cytometry. Plasma TNF activity was measured by L929 fibroblast cytolytic assay. Group Ps developed a significant neutropenia by 30 min (14.9 +/- 2.5 vs 23.4 +/- 3.3 x 10(3) cells/microliter at baseline, P less than 0.05, ANOVA) with circulating neutrophils exhibiting significantly increased CD18 expression by 60 min (6.34 +/- 0.72 vs 5.01 +/- 0.52 equivalent soluble fluorescence molecules (ESFM) x 10(3) at baseline, P less than 0.05, ANOVA). Group Ps demonstrated a significant increase in plasma TNF activity by 30 min (2.5 +/- 0.9 vs 0.7 +/- 0.3 U/ml at baseline). There was no significant change in PMN count, PMN CD18 expression, or plasma TNF activity in Group C. In complimentary in vitro studies, porcine PMNs stimulated with recombinant human TNF-alpha (n = 5) demonstrated a time- and dose-dependent increase in CD18 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Ibuprofen attenuates hypochlorous acid production from neutrophils in porcine acute lung injury

UNLABELLED Hypochlorous acid (HOCl), a neutrophil-generated oxidant, has been implicated in tissue destruction in sepsis-induced acute lung injury (ALI). Ibuprofen, a cyclooxygenase inhibitor, successfully attenuates many of the physiological derangements in ALI. The aim of this study was to examine the role of PMN hypochlorous acid in sepsis-induced ALI and evaluate the effect of ibuprofen therapy. Neutrophils from three groups of young (15-25 kg), anesthetized swine were studied: controls (C, n = 7) received 0.9% NaCl, septic animals (Ps, n = 8) received live Pseudomonas aeruginosa (5 x 10(8) CFU/ml at 0.3 ml/20 kg/min) for 60 min, and ibuprofen-treated animals (Ps + I, n = 6) received Ps plus ibuprofen 12.5 mg/kg administered at 0 and 120 min. Neutrophils were isolated from peripheral blood at 0, 60, and 300 min and the rate and total production of HOCl were assessed on the basis of the ability of the amino acid taurine to trap HOCl. RESULTS Septic (Ps) PMNs produce 32% more HOCl, P less than 0.01, at 300 min than at baseline which was associated with a marked increase in both extravascular lung water (6.44 +/- 0.8 ml/kg, t = 0 vs 16.03 +/- 2.6 ml/kg, t = 300, P less than 0.01) and bronchoalveolar protein content (115 +/- 13 micrograms/ml, t = 0 vs 633 +/- 104 micrograms/ml, t = 300, P less than 0.01). Ibuprofen significantly attenuated (P less than 0.05) HOCl production when compared to Ps, in conjunction with significantly (P less than 0.05) reduced levels of extravascular lung water and bronchoalveolar lavage protein.

Delayed cyclo-oxygenase blockade reduces the neutrophil respiratory burst and plasma tumor necrosis factor levels in sepsis-induced acute lung injury.

Ibuprofen pretreatment attenuates the enhanced neutrophil (PMN) respiratory burst and reduces increased plasma tumor necrosis factor (TNF) activity in porcine sepsis-induced acute lung injury (ALI). These septic responses have been linked to increased alveolar-capillary membrane (ACM) permeability. This study was designed to establish whether delayed ibuprofen treatment would have the same effect and to examine the relationship between PMN oxidant generation and TNF. Three groups of anesthetized, ventilated pigs (15-25 kg) were used. Group Ps received Pseudomonas aeruginosa (5 x 10(8) CFU/mL at 0.3 mL/20 kg/min) for one hour IV; The control group (Con) received 0.9% NaCl. Group D-Ibu received ibuprofen 12.5 mg/kg as a delayed bolus at 30 minutes and again at 120 minutes after Ps. Protein (BAL-P, microgram/mL) in harvested bronchoalveolar lavage fluid and extravascular lung water (EVLW, mL/kg) were used to estimate the integrity of the ACM. Superoxide anion (O2-) generation (ferricytochrome c reduction) from circulating PMNs and plasma TNF activity (L929 fibroblast bioassay) were measured. The EVLW increased significantly (p less than 0.05), as did BAL-P (p less than 0.01), in the P. aeruginosa-treated animals at 300 minutes. These increases were abolished in Group D-Ibu: EVLW, 6.6 +/- 1.0 baseline vs. 14.6 +/- 2.6 Ps 300 vs. 6.8 +/- 0.9 D-Ibu 300; BAL-P, 175 +/- 28 baseline vs. 984 +/- 186 Ps 300 vs. 284 +/- 42.8 D-Ibu 300. Both enhanced PMN oxidant activity and increased plasma TNF activity were significantly attenuated by delayed ibuprofen treatment. These data support the efficacy of the nonsteroidal anti-inflammatory drug, ibuprofen, when used after the onset of a septic stimulus.

Intravenous nitroglycerin-induced intracranial hypertension.

Nitroglycerin therapy can cause dose-related increases in intracranial pressure. Rare cases of neurologic sequelae attributed to nitroglycerin have appeared in the literature. We report such a case, in which symptoms completely resolved after cessation of nitroglycerin therapy. Widespread use of high-dose iv nitroglycerin makes knowledge of this effect important for all practitioners.

Combinet ibuprofen and monoclonal antibody to tumor necrosis factor-α attenuate hemodynamic dysfunction and sepsis-induced acute lung injury. Discussion

A number of key mediators are implicated in the pathophysiology of sepsis. In previous studies of a septic porcine model, ibuprofen pretreatment prevented the early but not the late rise in pulmonary vascular resistance index (PVRI) and the early but not the late fall in arterial PO2 (PaO2), whereas monoclonal antibody to tumor necrosis factor alpha (anti-TNF alpha) prevented the late but not the early rise in PVRI and the late but not the early fall in PaO2. This study examined the impact of pretreatment with combined ibuprofen and anti-TNF-alpha on the course of sepsis and acute lung injury (ALI) in pigs. Three groups were studied for 5 hours. Groups I (n = 9) and II (n = 5) received a 1-hour infusion of Pseudomonas aeruginosa. Group II received ibuprofen (12.5 mg/kg) and anti-TNF-alpha (5 mg/kg) before P. aeruginosa, and a further bolus of ibuprofen at 120 minutes. Group III (n = 11) received sterile saline. Group I demonstrated a significant (p < 0.05) rise in plasma TNF-alpha that was abolished in group II. The SVRI in group II did not change significantly from baseline through the study and the SVRI rose sharply in group I following onset of the infusion of P. aeruginosa, as did PVRI. There was no significant change in PVRI from baseline in group II, except for the final 60 minutes; PVRI in group II was significantly less than in group I throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ pulmonary vascular perfusion for improved recovery of pulmonary intravascular macrophages

The microcirculation contains mononuclear phagocytes, with features characteristic of macrophages, adhered to luminal capillary surfaces by intercellular adhesion plaques. These pulmonary intravascular macrophages may play an important role in regulating lung vascular tone and capillary permeability, and may modulate capillary endothelial cell growth and replication by the secretion of soluble mediators (i.e., arachidonate metabolites, cytokines). This study describes a technique which utilizes in situ lung perfusion to remove intravascular macrophages in large numbers from the microcirculation of porcine lung (n = 26). This technique yielded 3.8 +/- 0.5 x 10(8) (mean +/- SEM) mononuclear cells which were highly phagocytic toward particulate carbon (phagocytic index, 80 +/- 6%). Harvested mononuclear phagocytes reestablished intercellular adhesion plaques when placed on small vessel porcine pulmonary artery endothelial cell monolayers and exhibited histochemical characteristics typical of monocyte/macrophage lineage cells. Mononuclear cells obtained from lung microcirculation displayed size heterogeneity varying from 10.4 to 16.5 microns in diameter. Both large and small cell populations phagocytosed particulate carbon. Morphometric studies performed on collagenase-treated lung demonstrated that in situ perfusion removed significant numbers of intravascular macrophages in lung capillaries. The technique described permits the rapid removal of anchored mononuclear phagocytes from lung capillaries with minimal postmortem delay.

Hemodynamic effects of bradykinin antagonism in porcine gram-negative sepsis.

Activation of the kallikrein-kinin system in sepsis has long been recognized, but its role, beneficial or pathologic, has not been defined. Recently, however, specific bradykinin (BK) antagonists have become available and this study investigated the effects of a BK antagonist, NPC17731 (Scios-Nova) on systemic and pulmonary hemodynamics in a model of gram-negative sepsis. Anesthetized swine were studied for 5 h receiving a 1-h infusion of saline (controls, group 1, N = 8) or live Pseudomonas aeruginosa (septic, group 2, N = 8). Group 3 (treatment, N = 6) received NPC17731 (5 mg/kg initial bolus followed by 1 mg/kg hourly) just prior to the onset of sepsis. Group 2 animals showed a rapid decrease in systemic arterial pressure (SAP) from 30 min onward, and sustained significant hypotension from 2 h onward. In group 3, SAP fell similarly until 2 h then progressively rose, returning to baseline levels by 5 h. In contrast, cardiac index fell progressively from 3 h onward in groups 2 and 3. Systemic vascular resistance index (SVRI) fell significantly by 2 h in group 2 animals, recovering to baseline by 5 h. Group 3 showed a similar initial fall followed by a rebound increase in SVRI, which, at 5 h was significantly raised above the other groups. Group 2 developed significant, persistent pulmonary artery hypertension which was not reduced by NPC17731. The data imply a significant role for bradykinin in the pathogenesis of hypotension in septic shock in this model. Septic shock was reversed by a BK antagonist which increased peripheral resistance without affecting cardiac output.(ABSTRACT TRUNCATED AT 250 WORDS)

Ibuprofen prevents deterioration in static transpulmonary compliance and transalveolar protein flux in septic porcine acute lung injury.

UNLABELLED The effects of intravenous ibuprofen on measurements of pulmonary function and alveolar capillary membrane permeability to protein in sepsis-induced porcine acute lung injury (ALI) were studied. Young swine (15-25 kg) were anesthetized, cannulated, and ventilated (5 cm H2O PEEP, 0.5 FIO2, and 15 cc/kg tidal volume). Three groups were studied: septic animals (Ps, n = 10) received Pseudomonas aeruginosa for 1 hr IV, controls (C, n = 9) received 0.9% NaCl, and ibuprofen-treated septic animals (Ps + Ibu, n = 7) received ibuprofen 12.5 mg/kg at 0 and 120 min post Ps. Systemic (SAP) and pulmonary (PAP) arterial pressures, PaO2, cardiac index (CI), static lung compliance (CL), EVLW (thermal cardiogreen), and peripheral white blood cell counts (WBC) were measured. Bronchoalveolar lavage (BAL) was performed for protein and % neutrophil (%PMN) content. RESULTS Ps produced significant (p less than 0.05) decreases in CL, PaO2, SAP, CI, and peripheral WBC and increases in PAP, EVLW, BAL protein, and %PMNs vs. controls. Ibu prevented the early increase in PAP and attenuated the late increase in PAP and EVLW. Ibu also maintained PaO2, CL, BAL protein, and %PMNs in BAL at control levels, but exhibited no significant effect on peripheral leukopenia. These data strongly suggest that ibuprofen administered before and at 120 min after onset of Pseudomonas infusion improves lung compliance and affects neutrophil function sufficiently to significantly ameliorate many of the physiologic derangements in acute sepsis.

Increased pulmonary artery pressure in association with Raynaud's phenomenon

Pulmonary artery vasospasm is a hypothesized antecedent and etiologic factor in the pulmonary hypertension associated with progressive systemic sclerosis. Recent clinical data refute the existence of pulmonary vasospasm in a cohort of patients with scleroderma and normal baseline pulmonary artery pressure. A case of episodic pulmonary hypertension associated with a digital Raynauds response is reported, and a cardiac mechanism is hypothesized.