Nichelle Raj

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

Enhanced heat shock protein 70 expression alters proteasomal degradation of Iκb kinase in experimental acute respiratory distress syndrome

Objectives:Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-&kgr;B (NF-&kgr;B) signal transduction pathway. NF-&kgr;B activation requires dissociation/degradation of a bound inhibitor, I&kgr;B&agr;. I&kgr;B&agr; degradation requires phosphorylation by I&kgr;B kinase, ubiquitination by the SCF&bgr;-TrCP (Skp1/Cullin1/Fbox &bgr;-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-&kgr;B activation at multiple points in the I&kgr;B&agr; degradative pathway. Design:Laboratory investigation. Setting:University medical center research laboratory. Subjects:Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. Interventions:Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-&agr; and transfected. NF-&kgr;B was examined using molecular biological tools. Measurements and Main Results:Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-&kgr;B and attenuated phosphorylation of I&kgr;B&agr;. AdHSP treatment reduced, but did not eliminate, phosphorylation of the &bgr;-subunit of I&kgr;B kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the I&kgr;B kinase signalosome. This stabilized intermediary complexes containing I&kgr;B kinase components, I&kgr;B&agr;, and NF-&kgr;B. Cellular studies indicate that although ubiquitination of I&kgr;B&agr; was maintained, proteasomal degradation was impaired by an indirect mechanism. Conclusions:Treatment of sepsis-induced lung injury with AdHSP limits NF-&kgr;B activation. This results from stabilization of intermediary NF-&kgr;B/I&kgr;B&agr;/I&kgr;B kinase complexes in a way that impairs proteasomal degradation of I&kgr;B&agr;. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Competitive and noncompetitive inhibition of myocardial cytochrome C oxidase in sepsis

Sepsis is the most common cause of death in intensive care units worldwide. The basic pathophysiologic defect in sepsis, causing functional abnormalities in many organ systems, remains elusive. One potential cause is disruption of oxidative phosphorylation in mitochondria. Here, we report that oxidation of cytochrome c by myocardial cytochrome c oxidase, the terminal oxidase in the electron transport chain, is competitively inhibited early in experimental sepsis (cecal ligation with single or double 23-gauge puncture) in mice. In severe sepsis (cecal ligation and double puncture, 75% mortality at 48 h), inhibition becomes noncompetitive by 48 h. The development of noncompetitive inhibition is associated with a decrease in heme a,a3 content, which is the key active site in the functional subunit (I) and catalyzes the reduction of molecular oxygen. In addition, there are persistently decreased steady-state levels of subunit I mRNA and protein after cecal ligation and double puncture. Both loss of heme and loss of subunit I could explain the observed irreversible inhibition of cytochrome c oxidase. Noncompetitive inhibition of cytochrome c oxidase may interrupt oxidative phosphorylation, leading to sepsis-associated cardiac depression. Importantly, this abnormality may underlie sepsis-associated dysfunction in other organ systems.

Cecal ligation and double puncture impairs heat shock protein 70 (HSP-70) expression in the lungs of rats.

Induction of the heat shock response may improve outcome from pathophysiological disturbances. This improvement is associated with and believed to result from expression of heat shock protein (HSP)-70. Therefore, we examined the temporal expression of HSP-70 in an animal model of acute respiratory distress syndrome (ARDS) secondary to fecal peritonitis. Specifically, we hypothesize that sepsis in rats impairs pulmonary HSP-70 expression. ARDS was induced in adolescent rats via cecal ligation and double puncture (2CLP). Sham-operated animals served as controls. Lung tissue was collected 0, 3, 6, 16, 24, and 48 h after 2CLP and sham operation. Northern blot hybridization analysis was performed to detect steady-state HSP-70 messenger ribonucleic (mRNA) levels. HSP-70 protein levels were determined via immunoblotting and immunohistochemistry. Mortality after 2CLP was 50% at 24 h and 75% at 48 h. Northern blot hybridization analysis revealed no significant change in steady-state HSP-70 mRNA levels in lung at any time after 2CLP. HSP-70 steady-state mRNA levels increased after sham operation and was higher than values in 2CLP at 6, 16, and 24 h. HSP-70 protein levels did not change over time in either group. Thus, the expression of HSP-70 does not change after 2CLP. Although lack of an increase in protein levels may be adaptive after sham operation, it is not appropriate after 2CLP. Therefore, failed HSP-70 expression represents a form of pulmonary epithelial dysfunction that may contribute to lung injury in sepsis.

Sepsis-induced cholestasis, steatosis, hepatocellular injury, and impaired hepatocellular regeneration are enhanced in interleukin-6 -/- mice.

Objective:Hepatic dysfunction is an important but poorly understood component of sepsis. In severe sepsis, liver dysfunction is characterized by cholestasis, steatosis, hepatocellular injury, impaired regeneration, a decreased response to the cytokine interleukin-6, and high mortality. To determine whether loss of interleukin-6 activity caused hepatic dysfunction and mortality, we induced sepsis in wild-type (interleukin-6 +/+) and interleukin-6 knockout (interleukin-6 −/−) mice. We hypothesized that sepsis in interleukin-6 −/− mice would increase cholestasis, steatosis, hepatocellular injury, and mortality and impair hepatocyte regeneration. Design:Randomized prospective experimental study. Setting:University medical laboratory. Subjects:Male adolescent C57Bl6 interleukin-6 +/+ and interleukin-6 −/− mice. Interventions:Mild sepsis was induced using cecal ligation and single puncture (CLP). Severe, lethal sepsis was induced using cecal ligation and double puncture (2CLP). Some mice received recombinant human interleukin-6 at the time of CLP/2CLP. All animals were fluid resuscitated at the time of surgery and every 24 hrs thereafter. In survival cohorts, mortality at 16, 24, 48, and 72 hrs was recorded. In separate cohorts, surviving animals were killed at 24 and 48 hrs, and liver tissue was harvested. A separate cohort of mice received bromodeoxyuridine for detection of regeneration. Measurements and Main Results:2CLP was 100% fatal within the first 12 hrs in interleukin-6 −/− mice. Mortality from 2CLP in interleukin-6 +/+ mice before 24 hrs was nil but was 90% by 72 hrs. At 72 hrs, CLP was 40% fatal in interleukin-6 +/+ mice but 90% in interleukin-6 −/− mice. CLP induced cholestasis, steatosis, and hepatocellular injury in interleukin-6 −/−, but not interleukin-6 +/+, mice. Regeneration was absent following CLP in interleukin-6 −/− animals but occurred in interleukin-6 +/+ mice. Early administration of recombinant human interleukin-6 did not reverse abnormalities in interleukin-6 −/− mice. Conclusions:The absence of interleukin-6 is an important determinant of hepatic dysfunction and mortality in sepsis.

Intrahepatic nuclear factor-kappa B activity and alpha 1-acid glycoprotein transcription do not predict outcome after cecal ligation and puncture in the rat.

ObjectiveSepsis is the leading cause of death in critically ill surgical patients. Septic hepatic dysfunction, an important determinant of outcome, is poorly understood but includes inappropriate transcriptional down-regulation. This may be modulated by proinflammatory cytokines. We hypothesized that intrahepatic changes in tumor necrosis factor (TNF)/interleukin (IL)-1-linked processes, such as the activation of the p50 homodimeric and the p65/p50 heterodimeric isoforms of the transcription factor nuclear factor (NF)-&kgr;B or transcription of the acute phase reactant &agr;1-acid glycoprotein (AGP), would correlate with recovery from sepsis. DesignProspective experimental comparison of sham operation and nonlethal and lethal sepsis in male Sprague-Dawley rats. InterventionsNonlethal sepsis was induced by using single-puncture cecal ligation and puncture (CLP). Lethal sepsis was induced via double-puncture CLP. NF-&kgr;B DNA binding activity was determined by using electrophoretic mobility shift analysis with differentiation of p50/p50 and p50/p65 isoforms by using appropriate antibodies. AGP transcription was assessed with transcription elongation analysis, intrahepatic IL-1&bgr;, and TNF-&agr; abundance by using immunohistochemistry, and serum IL-1&bgr; was assessed by using ELISA. Main ResultsOverall NF-&kgr;B activity increased equivalently over time after both single- and double-puncture CLP, with a peak occurring 3 hrs after intervention. In single-puncture CLP, there was an increase in the binding of the p50 homodimer form over time. After double-puncture CLP, no such change was observed. AGP transcription was increased equivalently in both models. Intrahepatic IL-1&bgr; was detected 16 and 24 hrs after single-puncture CLP and 6 hrs after double-puncture CLP. After double-puncture CLP, intrahepatic TNF-&agr; was detected at 6, 16, and 24 hrs. Serum IL-1&bgr; was undetectable after both single- and double-puncture CLP. ConclusionsAlthough AGP transcription was similar in mild and fulminant sepsis, double-puncture CLP increased the binding activity of the p50 homodimer relative to binding of the p50/p65 NF-&kgr;B heterodimer. These results imply that transcriptional activity not linked to acute phase responses is an important determinant of outcome in sepsis.

Protection against sepsis-induced lung injury by selective inhibition of protein kinase C-δ (δ-PKC)

Inflammation and proinflammatory mediators are activators of δ‐PKC. In vitro, δ‐PKC regulates proinflammatory signaling in neutrophils and endothelial and epithelial cells, cells that can contribute to lung tissue damage associated with inflammation. In this study, a specific δ‐PKC TAT peptide inhibitor was used to test the hypothesis that inhibition of δ‐PKC would attenuate lung injury in an animal model of ARDS. Experimental ARDS was induced in rats via 2CLP, a model of polymicrobial sepsis. Following 2CLP surgery, the δ‐PKC TAT inhibitory peptide (2CLP+δ‐PKC TAT in PBS) or PBS (2CLP+PBS) was administered intratracheally. Controls consisted of SO, where animals underwent a laparotomy without 2CLP. Twenty‐four hours after SO or 2CLP, blood, BALF, and lung tissue were collected. 2CLP induced δ‐PKC phosphorylation in the lung within 24 h. Treatment with the δ‐PKC TAT inhibitory peptide significantly decreased pulmonary δ‐PKC phosphorylation, indicating effective inhibition of δ‐PKC activation. Plasma and BALF levels of the chemokines CINC‐1 and MIP‐2 were elevated in 2CLP + PBS rats as compared with SO rats. Treatment with δ‐PKC TAT reduced 2CLP‐induced elevations in chemokine levels in BALF and plasma, suggesting that δ‐PKC modulated chemokine expression. Most importantly, intratracheal administration of δ‐PKC TAT peptide significantly attenuated inflammatory cell infiltration, disruption of lung architecture, and pulmonary edema associated with 2CLP. Thus, δ‐PKC is an important regulator of proinflammatory events in the lung. Targeted inhibition of δ‐PKC exerted a lung‐protective effect 24 h after 2CLP.

Enhanced expression of 70-kilodalton heat shock protein limits cell division in a sepsis-induced model of acute respiratory distress syndrome

Objective:Fibrotic changes are initiated early in acute respiratory distress syndrome. This may involve overproliferation of alveolar type II cells. In an animal model of acute respiratory distress syndrome, we have shown that the administration of an adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) limited pathophysiological changes. We hypothesized that this improvement may be modulated, in part, by an early AdHSP-induced attenuation of alveolar type II cell proliferation. Design:Laboratory investigation. Setting:Hadassah-Hebrew University and University of Pennsylvania animal laboratories. Subjects:Sprague-Dawley Rats (250 g). Interventions:Lung injury was induced in male Sprague-Dawley rats via cecal ligation and double puncture. At the time of cecal ligation and double puncture, we injected phosphate-buffered saline, AdHSP, or AdGFP (an adenoviral vector expressing the marker green fluorescent protein) into the trachea. Rats then received subcutaneous bromodeoxyuridine. In separate experiments, A549 cells were incubated with medium, AdHSP, or AdGFP. Some cells were also stimulated with tumor necrosis factor-α. After 48 hrs, cytosolic and nuclear proteins from rat lungs or cell cultures were isolated. These were subjected to immunoblotting, immunoprecipitation, electrophoretic mobility shift assay, fluorescent immunohistochemistry, and Northern blot analysis. Measurements and Main Results:Alveolar type I cells were lost within 48 hrs of inducing acute respiratory distress syndrome. This was accompanied by alveolar type II cell proliferation. Treatment with AdHSP preserved alveolar type I cells and limited alveolar type II cell proliferation. Heat shock protein 70 prevented overexuberant cell division, in part, by inhibiting hyperphosphorylation of the regulatory retinoblastoma protein. This prevented retinoblastoma protein ubiquitination and degradation and, thus, stabilized the interaction of retinoblastoma protein with E2F1, a key cell division transcription factor. Conclusions:Heat shock protein 70-induced attenuation of cell proliferation may be a useful strategy for limiting lung injury when treating acute respiratory distress syndrome if consistent in later time points.

Adenoviral Vector Transfection into the Pulmonary Epithelium after Cecal Ligation and Puncture in Rats

BackgroundAdenoviral-targeted gene delivery to respiratory epithelium can augment production of specific proteins. Therefore, it may be valuable in treating the acute respiratory distress syndrome. The authors tested the hypothesis that adenoviral vector uptake after cecal ligation and double puncture in rats, an animal model of the acute respiratory distress syndrome, is higher than that observed in controls that did not undergo operation (“nonoperated”) or those that underwent a sham operation (“sham-operated”). MethodsAdenoviruses expressing green fluorescent protein or Lac-Z were delivered into the lungs of anesthetized rats via tracheal catheter. Animals were killed 24 or 48 h later. Histopathology and green fluorescent protein expression were examined using light of fluorescence microscopy. Cellular localization of Lac-Z was determined with electron microscopy or semithin sectioning. Viral receptor density and localization were determined using immunoblotting and immunohistochemistry. ResultsAfter cecal ligation and double puncture, rats were hypoxic and tachypneic. Alveoli were segmentally consolidated, contained proteinaceous debris and neutrophils, and had thickened septa. Administration of adenoviruses to rats that were sham-operated or underwent cecal ligation and double puncture resulted in high levels of marker protein expression in cells lining alveoli. Use of 3 × 1011 plaque-forming units instead of 3 × 1012 plaque-forming units resulted in similar levels of green fluorescent protein expression with negligible viral-mediated lymphocytic infiltration. Semithin section and electron microscopy revealed expression primarily localized to type II alveolar cells. Abundance of &agr;v&bgr;3 integrins and human coxsackie–adenovirus receptor (receptors that modulate viral attachment and internalization) was increased after cecal ligation and double puncture, predominantly in type II pneumocytes. ConclusionsCecal ligation and double puncture induces histologic and functional changes consistent with the acute respiratory distress syndrome, increases surface expression of viral receptors, and enhances adenoviral-mediated gene transfer.

Enhanced Hsp70 expression protects against acute lung injury by modulating apoptotic pathways.

The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis-induced lung injury. Sepsis was induced in rats via cecal ligation and double puncture (2CLP). At the time of 2CLP PBS, AdHSP or AdGFP (an adenoviral vector expressing green fluorescent protein) were injected into the tracheas of septic rats. 48 hours later, lungs were isolated. One lung was fixed for TUNEL staining and immunohistochemistry. The other was homogenized to isolate cytosolic and nuclear protein. Immunoblotting, gel filtration and co-immunoprecipitation were performed in these extracts. In separate experiments MLE-12 cells were incubated with medium, AdHSP or AdGFP. Cells were stimulated with TNFα. Cytosolic and nuclear proteins were isolated. These were subjected to immunoblotting, co- immunoprecipitation and a caspase-3 activity assay. TUNEL assay demonstrated that AdHSP reduced alveolar cell apoptosis. This was confirmed by immunohistochemical detection of caspase 3 abundance. In lung isolated from septic animals, immunoblotting, co-immunoprecipitation and gel filtration studies revealed an increase in cytoplasmic complexes containing caspases 3, 8 and 9. AdHSP disrupted these complexes. We propose that Hsp70 impairs apoptotic cellular pathways via interactions with caspases. Disruption of large complexes resulted in stabilization of lower molecular weight complexes, thereby, reducing nuclear caspase-3. Prevention of apoptosis in lung injury may preserve alveolar cells and aid in recovery.