Karen E. Welty-Wolf

The CO/HO system reverses inhibition of mitochondrial biogenesis and prevents murine doxorubicin cardiomyopathy

The clinical utility of anthracycline anticancer agents, especially doxorubicin, is limited by a progressive toxic cardiomyopathy linked to mitochondrial damage and cardiomyocyte apoptosis. Here we demonstrate that the post-doxorubicin mouse heart fails to upregulate the nuclear program for mitochondrial biogenesis and its associated intrinsic antiapoptosis proteins, leading to severe mitochondrial DNA (mtDNA) depletion, sarcomere destruction, apoptosis, necrosis, and excessive wall stress and fibrosis. Furthermore, we exploited recent evidence that mitochondrial biogenesis is regulated by the CO/heme oxygenase (CO/HO) system to ameliorate doxorubicin cardiomyopathy in mice. We found that the myocardial pathology was averted by periodic CO inhalation, which restored mitochondrial biogenesis and circumvented intrinsic apoptosis through caspase-3 and apoptosis-inducing factor. Moreover, CO simultaneously reversed doxorubicin-induced loss of DNA binding by GATA-4 and restored critical sarcomeric proteins. In isolated rat cardiac cells, HO-1 enzyme overexpression prevented doxorubicin-induced mtDNA depletion and apoptosis via activation of Akt1/PKB and guanylate cyclase, while HO-1 gene silencing exacerbated doxorubicin-induced mtDNA depletion and apoptosis. Thus doxorubicin disrupts cardiac mitochondrial biogenesis, which promotes intrinsic apoptosis, while CO/HO promotes mitochondrial biogenesis and opposes apoptosis, forestalling fibrosis and cardiomyopathy. These findings imply that the therapeutic index of anthracycline cancer chemotherapeutics can be improved by the protection of cardiac mitochondrial biogenesis.

Fungal infections in lung and heart-lung transplant recipients. Report of 9 cases and review of the literature.

We reviewed the pattern and incidence of fungal infections in patients undergoing lung and heart-lung transplantation at Duke University Medical Center from September 1992 until August 1995, and present here 9 illustrative cases. Of the 73 lung and heart-lung transplant recipients studied, 59 (81%) had positive fungal cultures at some point after transplantation. The cases presented here illustrate that lung transplant recipients are predisposed to a wide variety of fungal infections. The clinical pattern of these infections ranges from asymptomatic to rapidly progressive fatal disease. In addition to the reactivation of previous fungal infections and recent exposure to new environmental sources, the donor lung itself can be the source of fungal infection, as we showed by using molecular epidemiology techniques. Because of the associated morbidity and mortality, efforts should be directed at investigating prophylactic antifungal regimens in lung transplant recipients. Preliminary reports on the use of itraconazole and aerosolized amphotericin B have been encouraging. Prospective randomized studies are needed to assess the safety and cost effectiveness of different regimens. Fungal infections in patients after lung transplantation can significantly impede recovery and lead to substantial mortality.

Lipopolysaccharide Stimulates Mitochondrial Biogenesis via Activation of Nuclear Respiratory Factor-1

Exposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase γ, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (ρo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage.

Heme Oxygenase-1 Couples Activation of Mitochondrial Biogenesis to Anti-inflammatory Cytokine Expression

The induction of heme oxygenase-1 (HO-1; Hmox1) by inflammation, for instance in sepsis, is associated both with an anti-inflammatory response and with mitochondrial biogenesis. Here, we tested the idea that HO-1, acting through the Nfe2l2 (Nrf2) transcription factor, links anti-inflammatory cytokine expression to activation of mitochondrial biogenesis. HO-1 induction after LPS stimulated anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1Ra) expression in mouse liver, human HepG2 cells, and mouse J774.1 macrophages but blunted tumor necrosis factor-α expression. This was accompanied by nuclear Nfe2l2 accumulation and led us to identify abundant Nfe2l2 and other mitochondrial biogenesis transcription factor binding sites in the promoter regions of IL10 and IL1Ra compared with pro-inflammatory genes regulated by NF-κΒ. Mechanistically, HO-1, through its CO product, enabled these transcription factors to bind the core IL10 and IL1Ra promoters, which for IL10 included Nfe2l2, nuclear respiratory factor (NRF)-2 (Gabpa), and MEF2, and for IL1Ra, included NRF-1 and MEF2. In cells, Hmox1 or Nfe2l2 RNA silencing prevented IL-10 and IL-1Ra up-regulation, and HO-1 induction failed post-LPS in Nfe2l2-silenced cells and post-sepsis in Nfe2l2−/− mice. Nfe2l2−/− mice compared with WT mice, showed more liver damage, higher mortality, and ineffective CO rescue in sepsis. Nfe2l2−/− mice in sepsis also generated higher hepatic TNF-α mRNA levels, lower NRF-1 and PGC-1α mRNA levels, and no enhancement of anti-inflammatory Il10, Socs3, or bcl-xL gene expression. These findings disclose a highly structured transcriptional network that couples mitochondrial biogenesis to counter-inflammation with major implications for immune suppression in sepsis.

Activation of Mitochondrial Biogenesis by Heme Oxygenase-1–mediated NF-E2–related Factor-2 Induction Rescues Mice from Lethal Staphylococcus aureus Sepsis

RATIONALE Mitochondrial damage is an important component of multiple organ failure syndrome, a highly lethal complication of severe sepsis that lacks specific therapy. Mitochondrial quality control is regulated in part by the heme oxygenase-1 (HO-1; Hmox1) system through the redox-regulated NF-E2-related factor-2 (Nrf2) transcription factor, but its role in mitochondrial biogenesis in Staphylococcus aureus sepsis is unknown. OBJECTIVES To test the hypothesis that Nrf2-dependent up-regulation of the HO-1/carbon monoxide (CO) system would preserve mitochondrial biogenesis and rescue mice from lethal S. aureus sepsis. METHODS A controlled murine S. aureus peritonitis model with and without inhaled CO was examined for HO-1 and Nrf2 regulation of mitochondrial biogenesis and the resolution of hepatic mitochondrial damage. MEASUREMENTS AND MAIN RESULTS Sepsis survival was significantly enhanced using inhaled CO (250 ppm once-daily for 1 h), and linked mechanistically to Hmox1 induction and mitochondrial HO activity through Nrf2 transcriptional and Akt kinase activity. HO-1/CO stimulated Nrf2-dependent gene expression and nuclear accumulation of nuclear respiratory factor-1, -2α (Gabpa), and peroxisome proliferator-activated receptor gamma coactivator-1α; increased mitochondrial transcription factor-A and citrate synthase protein levels; and augmented mtDNA copy number. CO enhanced antiinflammatory IL-10 and reduced proinflammatory tumor necrosis factor-α production. By contrast, Nrf2(-/-) and Akt1(-/-) mice lacked CO induction of Hmox1 and mitochondrial biogenesis, and CO rescued neither strain from S. aureus sepsis. CONCLUSIONS We identify an inducible Nrf2/HO-1 regulatory cycle for mitochondrial biogenesis that is prosurvival and counter-inflammatory in sepsis, and describe targeted induction of mitochondrial biogenesis as a potential multiple organ failure therapy.

Toll-like receptor 4 mediates mitochondrial DNA damage and biogenic responses after heat-inactivated E. coli

An important site of cellular damage in bacterial sepsis is mitochondrial DNA (mtDNA), which we proposed is caused by reactive oxygen and nitrogen species generated by activation of signaling through specific toll‐like receptors (TLR). In wild‐type (Wt) mice injected with heat‐inactivated E. coli, hepatic TLR4 and TLR2 proteins were up‐regulated with TLR‐dependent increases in transcript levels for tumor necrosis factor (TNF‐α), interleukin 6, nitric oxide synthase‐II (iNOS), and NADPH oxidase 2 (Nox2). The accompanying stress significantly depleted hepatic mtDNA despite eight‐ and fourfold increases in manganese superoxide dismutase (MnSOD) and mitochondrial transcription factor A (Tfam) expression, respectively. The identical E. coli dose generated significantly less TNF‐α, NO, and Nox2 in TLR4−/− and TLR2/4−/− but not in TLR2−/− mice. TLR4−/− and TLR2/4−/− compared with Wt mice were protected from mtDNA oxidation but showed no Tfam up‐regulation and little copy number restoration. A critical role in the mtDNA damage was determined for TLR4‐mediated iNOS transcription through the MyD88 pathway. In Wt mice, mtDNA depletion was avoided by selective iNOS blockade, and residual mtDNA loss was linked to NF‐κB‐dependent TNF‐α expression. These data disclose the dual role of TLR4 in mtDNA damage and compensatory mitochondrial biogenic responses after innate immune activation.

Nrf2 promotes alveolar mitochondrial biogenesis and resolution of lung injury in Staphylococcus aureus pneumonia in mice

Acute lung injury (ALI) initiates protective responses involving genes downstream of the Nrf2 (Nfe2l2) transcription factor, including heme oxygenase-1 (HO-1), which stimulates mitochondrial biogenesis and related anti-inflammatory processes. We examined mitochondrial biogenesis during Staphylococcus aureus pneumonia in mice and the effect of Nrf2 deficiency on lung mitochondrial biogenesis and resolution of lung inflammation. S. aureus pneumonia established by nasal insufflation of live bacteria was studied in mitochondrial reporter (mt-COX8-GFP) mice, wild-type (WT) mice, and Nrf2⁻/⁻ mice. Bronchoalveolar lavage, wet/dry ratios, real-time RT-PCR and Western analysis, immunohistochemistry, and fluorescence microscopy were performed on the lung at 0, 6, 24, and 48 h. The mice survived S. aureus inoculations at 5×10⁸ CFU despite diffuse lung inflammation and edema, but the Nrf2⁻/⁻ lung showed increased ALI. In mt-COX8-GFP mice, mitochondrial fluorescence was enhanced in bronchial and alveolar type II (AT2) epithelial cells. WT mice displayed rapid HO-1 upregulation and lower proinflammatory TNF-α, IL-1β, and CCL2 and, especially in AT2 cells, higher anti-inflammatory IL-10 and suppressor of cytokine signaling-3 than Nrf2⁻/⁻ mice. In the alveolar region, WT but not Nrf2⁻/⁻ mice showed strongly induced nuclear respiratory factor-1, PGC-1α, mitochondrial transcription factor-A, SOD2, Bnip3, mtDNA copy number, and citrate synthase. These findings indicate that S. aureus pneumonia induces Nrf2-dependent mitochondrial biogenesis in the alveolar region, mainly in AT2 cells. Absence of Nrf2 suppresses the alveolar transcriptional network for mitochondrial biogenesis and anti-inflammation, which worsens ALI. The findings link redox activation of mitochondrial biogenesis to ALI resolution.

Ultrastructural changes in skeletal muscle mitochondria in gram-negative sepsis.

Energy metabolism during sepsis is incompletely understood, but alterations in mitochondrial structure and function appear important. We measured time-dependent changes in mitochondrial structure during sepsis using serial skeletal muscle biopsies in anesthetized baboons injected with 1010CFU/kg of live Escherichia coli (LD100). Skeletal muscle biopsies were taken before bacterial challenge (0 h controls) and at 12 h, 24 h, and death. By qualitative electron microscopy, the organelles became enlarged with distorted cristae and developed electron lucent areas within the matrix. With advanced injury the inner membrane became fragmented. Quantitative morphometric analysis showed a 50% increase in mean cristal membrane surface density by 24 h (p<.05) accompanied by a 100% increase in intermembrane space (p<.01). Matrix volume density decreased progressively (p<.01). These changes in mitochondrial ultrastructure occur within 12 h after the onset of the bacterial insult. This damage, including destruction or reorganization of both membrane and matrix proteins, is severe enough to compromise oxidative metabolism in muscle in Gram-negative sepsis.

Nitric oxide synthase-2 induction optimizes cardiac mitochondrial biogenesis after endotoxemia

Mitochondrial biogenesis protects metabolism from mitochondrial dysfunction produced by activation of innate immunity by lipopolysaccharide (LPS) or other bacterial products. Here we tested the hypothesis in mouse heart that activation of toll-like receptor-4 (TLR4), which induces early-phase genes that damage mitochondria, also activates mitochondrial biogenesis through induction of nitric oxide synthase (NOS2). We compared three strains of mice: wild type (Wt) C57BL/6J, TLR4(-/-), and NOS2(-/-)for cardiac mitochondrial damage and mitochondrial biogenesis by real-time RT-PCR, Western analysis, immunochemistry, and isoform analysis of myosin heavy chain (MHC) after sublethal heat-killed Escherichia coli (HkEC). After HkEC, Wt mice displayed significant myocardial mtDNA depletion along with enhanced TLR4 and NOS2 gene and protein expression that normalized in 72 h. HkEC generated less cytokine stress in TLR4(-/-)and NOS2(-/-)than Wt mice, NOS2(-/-)mice had mtDNA damage comparable to Wt, and both knockout strains failed to restore mtDNA copy number because of mitochondrial transcriptosome dysfunction. Wt mice also showed the largest beta-MHC isoform switch, but MHC recovery lagged in the NOS2(-/-)and TLR4(-/-)strains. The NOS2(-/-)mice also unexpectedly revealed the codependency of TLR4 expression on NOS2. These findings demonstrate the decisive participation of NOS2 induction by TLR4 in optimization of mitochondrial biogenesis and MHC expression after gram-negative challenge.

Tissue factor in experimental acute lung injury

Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.