Apoptosis Repressor With Caspase Recruitment Domain Ameliorates Amyloid-Induced ß-Cell Apoptosis and JNK Pathway Activation.

Islet amyloid is present in more than 90% of individuals with type 2 diabetes, where it contributes to ß-cell apoptosis and insufficient insulin secretion. Apoptosis repressor with caspase recruitment domain (ARC) binds and inactivates components of the intrinsic and extrinsic apoptosis pathways and was recently found to be expressed in islet ß-cells. Using a human islet amyloid polypeptide transgenic mouse model of islet amyloidosis, we show ARC knockdown increases amyloid-induced ß-cell apoptosis and loss, while ARC overexpression decreases amyloid-induced apoptosis, thus preserving ß-cells. These effects occurred in the absence of changes in islet amyloid deposition, indicating ARC acts downstream of amyloid formation. Because islet amyloid increases c-Jun N-terminal kinase (JNK) pathway activation, we investigated whether ARC affects JNK signaling in amyloid-forming islets. We found ARC knockdown enhances JNK pathway activation, whereas ARC overexpression reduces JNK, c-Jun phosphorylation, and c-Jun target gene expression (Jun and Tnf). Immunoprecipitation of ARC from mouse islet lysates showed ARC binds JNK, suggesting interaction between JNK and ARC decreases amyloid-induced JNK phosphorylation and downstream signaling. These data indicate that ARC overexpression diminishes amyloid-induced JNK pathway activation and apoptosis in the ß-cell, a strategy that may reduce ß-cell loss in type 2 diabetes.

The aquaporin 1 C-terminal tail is required for migration and growth of pulmonary arterial myocytes.

Pulmonary arterial smooth muscle cell (PASMC) proliferation and migration are important contributors to the vascular remodeling that occurs during development of pulmonary hypertension. We previously demonstrated that aquaporin (AQP)1, a member of the water channel family of proteins, was expressed in PASMCs and was necessary for hypoxia-induced migration; however, the mechanism by which AQP1 controls this response is unclear. The C-terminal tail of AQP1 contains putative calcium (EF-hand) and protein binding sites. Thus, we wanted to explore whether the C-terminal tail or the EF-hand motif of AQP1 was required for migration and proliferation. Rat PASMCs were isolated from distal pulmonary arteries, and proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. To deplete AQP1, PASMCs were transfected with AQP1 small interference RNA (siRNA) or nontargeting siRNA. Knockdown of AQP1 reduced basal proliferation and hypoxia-induced migration and proliferation in PASMCs. In subsequent experiments, wild-type AQP1, AQP1 lacking the entire cytoplasmic C-terminal tail, or AQP1 with a mutation in the EF-hand motif were expressed in PASMCs using adenoviral constructs. For all AQP1 constructs, infection increased AQP1 protein levels, water permeability, and the change in cell volume induced by hypotonic challenge. Infection with wild-type and EF-hand mutated AQP1, but not C-terminal-deleted AQP1, increased PASMC migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs and that the mechanism requires the C-terminal tail of the protein but is independent of water transport or the EF-hand motif.

Mitogen-activated protein kinase-activated protein kinase 2 mediates apoptosis during lung vascular permeability by regulating movement of cleaved caspase 3.

Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPK's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.

The apoptosis inhibitor ARC alleviates the ER stress response to promote ß-cell survival.

Type 2 diabetes involves insulin resistance and ß-cell failure leading to inadequate insulin secretion. An important component of ß-cell failure is cell loss by apoptosis. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of apoptosis that is expressed in cardiac and skeletal myocytes and neurons. ARC possesses the unusual property of antagonizing both the extrinsic (death receptor) and intrinsic (mitochondria/endoplasmic reticulum [ER]) cell death pathways. Here we report that ARC protein is abundant in cells of the endocrine pancreas, including >99.5% of mouse and 73% of human ß-cells. Using genetic gain- and loss-of-function approaches, our data demonstrate that ARC inhibits ß-cell apoptosis elicited by multiple inducers of cell death, including ER stressors tunicamycin, thapsigargin, and physiological concentrations of palmitate. Unexpectedly, ARC diminishes the ER stress response, acting distal to protein kinase RNA-like ER kinase (PERK) and inositol-requiring protein 1α, to suppress C/EBP homologous protein (CHOP) induction. Depletion of ARC in isolated islets augments palmitate-induced apoptosis, which is dramatically rescued by deletion of CHOP. These data demonstrate that ARC is a previously unrecognized inhibitor of apoptosis in ß-cells and that its protective effects are mediated through suppression of the ER stress response pathway.

Resolution of experimental lung injury by monocyte-derived inducible nitric oxide synthase.

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS-/- mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS-/- mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS-/- mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS-/- mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS-/- mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.

Bax regulates primary necrosis through mitochondrial dynamics.

The defining event in apoptosis is mitochondrial outer membrane permeabilization (MOMP), allowing apoptogen release. In contrast, the triggering event in primary necrosis is early opening of the inner membrane mitochondrial permeability transition pore (mPTP), precipitating mitochondrial dysfunction and cessation of ATP synthesis. Bcl-2 proteins Bax and Bak are the principal activators of MOMP and apoptosis. Unexpectedly, we find that deletion of Bax and Bak dramatically reduces necrotic injury during myocardial infarction in vivo. Triple knockout mice lacking Bax/Bak and cyclophilin D, a key regulator of necrosis, fail to show further reduction in infarct size over those deficient in Bax/Bak. Absence of Bax/Bak renders cells resistant to mPTP opening and necrosis, effects confirmed in isolated mitochondria. Reconstitution of these cells or mitochondria with wild-type Bax, or an oligomerization-deficient mutant that cannot support MOMP and apoptosis, restores mPTP opening and necrosis, implicating distinct mechanisms for Bax-regulated necrosis and apoptosis. Both forms of Bax restore mitochondrial fusion in Bax/Bak-null cells, which otherwise exhibit fragmented mitochondria. Cells lacking mitofusin 2 (Mfn2), which exhibit similar fusion defects, are protected to the same extent as Bax/Bak-null cells. Conversely, restoration of fused mitochondria through inhibition of fission potentiates mPTP opening in the absence of Bax/Bak or Mfn2, indicating that the fused state itself is critical. These data demonstrate that Bax-driven fusion lowers the threshold for mPTP opening and necrosis. Thus, Bax and Bak play wider roles in cell death than previously appreciated and may be optimal therapeutic targets for diseases that involve both forms of cell death.

A critical role for muscle ring finger-1 in acute lung injury-associated skeletal muscle wasting.

RATIONALE: Acute lung injury (ALI) is a debilitating condition associated with severe skeletal muscle weakness that persists in humans long after lung injury has resolved. The molecular mechanisms underlying this condition are unknown. OBJECTIVES: To identify the muscle-specific molecular mechanisms responsible for muscle wasting in a mouse model of ALI. METHODS: Changes in skeletal muscle weight, fiber size, in vivo contractile performance, and expression of mRNAs and proteins encoding muscle atrophy-associated genes for muscle ring finger-1 (MuRF1) and atrogin1 were measured. Genetic inactivation of MuRF1 or electroporation-mediated transduction of miRNA-based short hairpin RNAs targeting either MuRF1 or atrogin1 were used to identify their role in ALI-associated skeletal muscle wasting. MEASUREMENTS AND MAIN RESULTS: Mice with ALI developed profound muscle atrophy and preferential loss of muscle contractile proteins associated with reduced muscle function in vivo. Although mRNA expression of the muscle-specific ubiquitin ligases, MuRF1 and atrogin1, was increased in ALI mice, only MuRF1 protein levels were up-regulated. Consistent with these changes, suppression of MuRF1 by genetic or biochemical approaches prevented muscle fiber atrophy, whereas suppression of atrogin1 expression was without effect. Despite resolution of lung injury and down-regulation of MuRF1 and atrogin1, force generation in ALI mice remained suppressed. CONCLUSIONS: These data show that MuRF1 is responsible for mediating muscle atrophy that occurs during the period of active lung injury in ALI mice and that, as in humans, skeletal muscle dysfunction persists despite resolution of lung injury.

A critical role for the protein apoptosis repressor with caspase recruitment domain in hypoxia-induced pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a lethal syndrome associated with the pathogenic remodeling of the pulmonary vasculature and the emergence of apoptosis-resistant cells. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of multiple forms of cell death known to be abundantly expressed in striated muscle. We show for the first time that ARC is expressed in arterial smooth muscle cells of the pulmonary vasculature and is markedly upregulated in several experimental models of PH. In this study, we test the hypothesis that ARC expression is essential for the development of chronic hypoxia-induced PH. METHODS AND RESULTS: Experiments in which cells or mice were rendered ARC-deficient revealed that ARC not only protected pulmonary arterial smooth muscle cells from hypoxia-induced death, but also facilitated growth factor-induced proliferation and hypertrophy and hypoxia-induced downregulation of selective voltage-gated potassium channels, the latter a hallmark of the syndrome in humans. Moreover, ARC-deficient mice exhibited diminished vascular remodeling, increased apoptosis, and decreased proliferation in response to chronic hypoxia, resulting in marked protection from PH in vivo. Patients with PH have significantly increased ARC expression not only in remodeled vessels but also in the lumen-occluding lesions associated with severe disease. CONCLUSIONS: These data show that ARC, previously unlinked to pulmonary hypertension, is a critical determinant of vascular remodeling in this syndrome.

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury.

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation. Compared with IT water controls, LPS alone (3.75 µg/g body wt) increased lung PDE2A mRNA and protein expression by 6 h with a persistent increase in protein through day 4 before decreasing to control levels on days 6 and 10. Similar to the PDE2A time course, the peak in bronchoalveolar lavage (BAL) neutrophils, lactate dehydrogenase (LDH), and protein concentration also occurred on day 4 post-LPS. IT LPS (1 day) and VILI caused a threefold increase in lung PDE2A and inducible nitric oxide synthase (iNOS) and a 24-fold increase in BAL neutrophilia. Compared with a control adenovirus, PDE2A knockdown with an adenovirus expressing a short hairpin RNA administered IT 3 days before LPS/VILI effectively decreased lung PDE2A expression and significantly attenuated BAL neutrophilia, LDH, protein, and chemokine levels. PDE2A knockdown also reduced lung iNOS expression by 53%, increased lung cAMP by nearly twofold, and improved survival from 47 to 100%. We conclude that in a mouse model of LPS/VILI, a synergistic increase in lung PDE2A expression increased lung iNOS and alveolar inflammation and contributed significantly to the ensuing acute lung injury.

Induction of the apoptosis inhibitor ARC by Ras in human cancers.

Inhibition of apoptosis is critical for carcinogenesis. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both intrinsic and extrinsic apoptosis pathways. Although normally expressed in striated myocytes and neurons, ARC is markedly induced in a variety of primary human epithelial cancers and renders cancer cells resistant to killing. The mechanisms that mediate the induction of ARC in cancer are unknown. Herein we demonstrate that increases in ARC abundance are stimulated by Ras through effects on transcription and protein stability. Overexpression of activated N-Ras or H-Ras in normal cells is sufficient to increase ARC mRNA and protein levels. Similarly, transgenic expression of activated H-Ras induces ARC in both the normal mammary epithelium and resulting tumors of intact mice. Conversely, knockdown of endogenous N-Ras in breast and colon cancer cells significantly reduces ARC mRNA and protein levels. The promoter of the Nol3 locus, encoding ARC, is activated by N-Ras and H-Ras in a MEK/ERK-dependent manner. Ras also stabilizes ARC protein by suppressing its polyubiquitination and subsequent proteasomal degradation. In addition to the effects of Ras on ARC abundance, ARC mediates Ras-induced cell survival and cell cycle progression. Thus, Ras induces ARC in epithelial cancers, and ARC plays a role in the oncogenic actions of Ras.

Mycophenolate mofetil modifies kidney tubular injury and Foxp3+ regulatory T cell trafficking during recovery from experimental ischemia-reperfusion.

Lymphocytes participate in the early pathogenesis of ischemia-reperfusion injury (IRI) in kidney; however, their role during repair is largely unknown. Recent data have shown that Foxp3(+) regulatory T cells (Tregs) traffic into kidney during healing from IRI and directly participate in repair. Since lymphocyte-targeting therapy is currently administered to prevent rejection during recovery from IRI in renal transplants, we hypothesized that mycophenolate mofetil (MMF) would alter Treg trafficking and kidney repair. C57BL/6J and T cell deficient mice underwent unilateral clamping of renal pedicle for 45 min, followed by reperfusion, and were sacrificed at day 10. Mice were treated with saline (C) or MMF (100mg/kg) i.p. daily starting at day 2 until sacrifice (n=5-12/group). MMF worsened kidney tubular damage compared to C at 10 days (cortex and outer medulla: p<0.05) in wild-type mice; tubular apoptotic index was increased in cortex in MMF group as well (p=0.01). MMF reduced the total number of kidney-infiltrating mononuclear cells (p<0.001 versus C) and the percentages of TCRbeta(+)CD4(+) and TCRbeta(+)CD8(+) T cells (p<0.01), but not natural killer (NK), NKT or B lymphocytes. MMF specifically reduced kidney Foxp3(+) Tregs (0.82+/-0.11% versus 1.75+/-0.17%, p<0.05). Tubular proliferative index and tissue levels of basic FGF were increased in MMF group (p<0.05), IL-10 and IL-6 were decreased (p<0.05). To evaluate if MMF effect occurred through non-lymphocytic cells, T cell deficient mice were treated with MMF. Tubular injury in T cell deficient mice was not affected by MMF treatment, though MMF-treated animals had increased VEGF and decreased PDGF-BB protein tissue levels compared to controls (p<0.05). Thus, MMF modifies the structural, epithelial proliferative and inflammatory response during healing, likely through effects on T cells and possibly Tregs. Kidney repair after IRI can be altered by agents that target lymphocytes.

Foxp3+ regulatory T cells participate in repair of ischemic acute kidney injury.

T lymphocytes modulate early ischemia-reperfusion injury in the kidney; however, their role during repair is unknown. We studied the role of TCRbeta(+)CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), known to blunt immune responses, in repair after ischemia-reperfusion injury to the kidney. Using a murine model of ischemic acute kidney injury we found that there was a significant trafficking of Tregs into the kidneys after 3 and 10 days. Post-ischemic kidneys had increased numbers of TCRbeta(+)CD4(+) and TCRbeta(+)CD8(+) T cells with enhanced pro-inflammatory cytokine production. Treg depletion starting 1 day after ischemic injury using anti-CD25 antibodies increased renal tubular damage, reduced tubular proliferation at both time points, enhanced infiltrating T lymphocyte cytokine production at 3 days and TNF-alpha generation by TCRbeta(+)CD4(+) T cells at 10 days. In separate mice, infusion of CD4(+)CD25(+) Tregs 1 day after initial injury reduced INF-gamma production by TCRbeta(+)CD4(+) T cells at 3 days, improved repair and reduced cytokine generation at 10 days. Treg manipulation had minimal effect on neutrophil and macrophage infiltration; Treg depletion worsened mortality and serum creatinine, while Treg infusion had a late beneficial effect on serum creatinine in bilateral ischemia. Our study demonstrates that Tregs infiltrate ischemic-reperfused kidneys during the healing process promoting repair, likely through modulation of pro-inflammatory cytokine production of other T cell subsets. Treg targeting could be a novel therapeutic approach to enhance recovery from ischemic acute kidney injury.

Pulmonary epithelial neuropilin-1 deletion enhances development of cigarette smoke-induced emphysema.

RATIONALE: Cigarette smoke (CS) exposure is an important risk factor for chronic obstructive pulmonary disease; however, not all smokers develop disease, suggesting that other factors influence disease development. OBJECTIVES: We sought to determine whether neuropilin-1 (Nrp1), an integral component of receptor complexes mediating alveolar septation and vascular development, was involved in maintenance of normal alveolar structure, and/or altered susceptibility to the effects of CS. METHODS: Transgenic mice were generated to achieve inducible lung-specific deletion of epithelial Nrp1. We determined whether conditional Nrp1 deletion altered airspace size, then compared the effects of chronic CS or filtered air exposure on airspace size, inflammation, and the balance between cell death and proliferation in conditionally Nrp1-deficient adult mice and littermate controls. Finally, we evaluated the effects of Nrp1 silencing on cell death after acute exposure of A549 cells to cigarette smoke extract or short chain ceramides. MEASUREMENTS AND MAIN RESULTS: Genetic deletion of epithelial Nrp1 in either postnatal or adult lungs resulted in a small increase in airspace size. More notably, both airspace enlargement and apoptosis of type I and type II alveolar epithelial cells were significantly enhanced following chronic CS exposure in conditionally Nrp1-deficient adult mice. Silencing of Nrp1 in A549 cells did not alter cell survival after vehicle treatment but significantly augmented apoptosis after exposure to cigarette smoke extract or ceramide. CONCLUSIONS: These data support a role for epithelial Nrp1 in the maintenance of normal alveolar structure and suggest that dysregulation of Nrp1 expression may promote epithelial cell death in response to CS exposure, thereby enhancing emphysema development.

Transcription factor Nrf2 is protective during ischemic and nephrotoxic acute kidney injury in mice.

Oxidative stress is involved in acute kidney injury due to ischemia-reperfusion and chemotherapy-induced nephrotoxicity. To investigate their basic mechanisms we studied the role of nuclear factor-erythroid 2-p45-related factor 2 (Nrf2), a redox-sensitive transcription factor that regulates expression of several antioxidant and cytoprotective genes. We compared the responses of Nrf2-knockout mice and their wild-type littermates in established mouse models of ischemia-reperfusion injury and cisplatin-induced nephrotoxicity. Several Nrf2-regulated genes encoding antioxidant enzymes/proteins were significantly upregulated in the kidneys of wild type but not Nrf2-knockout mice following renal ischemia. Renal function, histology, vascular permeability, and survival were each significantly worse in the Nrf2 knockout mice. Further, proinflammatory cytokine and chemokine expression tended to increase after ischemia in the knockout compared to the wild-type mice. Treatment of the knockout mice with the antioxidants N-acetyl-cysteine or glutathione improved renal function. The knockout mice were more susceptible to cisplatin-induced nephrotoxicity, and this was blunted by N-acetyl-cysteine pretreatment. Our study demonstrates that Nrf2-deficiency enhances susceptibility to both ischemic and nephrotoxic acute kidney injury, and identifies this transcription factor as a potential therapeutic target in these injuries.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) induces the vascular and hemodynamic changes of pulmonary hypertension.

Pulmonary hypertension (PH) is a serious disease of multiple etiologies mediated by hypoxia, immune stimuli, and elevated pulmonary pressure that leads to vascular thickening and eventual right heart failure. In a chronic hypoxia model of PH, we previously reported the induction of a novel pleiotropic cytokine, hypoxia-induced mitogenic factor (HIMF), that exhibits mitogenic, vasculogenic, contractile, and chemokine properties during PH-associated vascular remodeling. To examine the role of HIMF in hypoxia-induced vascular remodeling, we performed in vivo knockdown of HIMF using short hairpin RNA directed at rat HIMF in the chronic hypoxia model of PH. Knockdown of HIMF partially blocked increases in mean pulmonary artery pressure, pulmonary vascular resistance, right heart hypertrophy, and vascular remodeling caused by chronic hypoxia. To demonstrate a direct role for HIMF in the mechanism of PH development, we performed HIMF-gene transfer into the lungs of rats using a HIMF-expressing adeno-associated virus (AAV). AAV-HIMF alone caused development of PH similar to that of chronic hypoxia with increased mean pulmonary artery pressure and pulmonary vascular resistance, right heart hypertrophy, and neomuscularization and thickening of small pulmonary arterioles. The findings suggest that HIMF represents a critical cytokine-like growth factor in the development of PH.

Sphingosine 1-phosphate rescues canine LPS-induced acute lung injury and alters systemic inflammatory cytokine production in vivo.

S1P has been demonstrated to protect against the formation of lipopolysaccharide (LPS)-induced lung edema when administered concomitantly with LPS. In the current study, we sought to determine the effectiveness of S1P to attenuate lung injury in a translationally relevant canine model of ALI when administered as rescue therapy. Secondarily, we examined whether the attenuation of LPS-induced physiologic lung injury after administration of S1P was, at least in part, caused by an alteration in local and/or systemic inflammatory cytokine expression. We examined 18, 1-year-old male beagles prospectively in which we instilled bacterial LPS (2-4 mg/kg) intratracheally followed in 1 h with intravenous S1P (85 microg/kg) or vehicle and 8 h of high-tidal-volume mechanical ventilation. S1P attenuated the formation of Q(s)/Q(t) (32%), and both the presence of protein (72%) and neutrophils (95%) in BAL fluid compared with vehicle controls. Although lung tissue inflammatory cytokine production was found to vary regionally throughout the LPS-injured lung, S1P did not alter the expression pattern. Similarly, BAL cytokine production was not altered significantly by intravenous S1P in this model. Interestingly, S1P potentiated the LPS-induced systemic production of 3 inflammatory cytokines, TNF-alpha (6-fold), KC (1.2-fold), and IL-6 (3-fold), without resulting in end-organ dysfunction. In conclusion, intravenous S1P reduces inflammatory lung injury when administered as rescue therapy in our canine model of LPS-induced ALI. This improvement is observed in the absence of changes in local pulmonary inflammatory cytokine production and an augmentation of systemic inflammation.

Expression, activity, and pro-hypertrophic effects of PDE5A in cardiac myocytes.

Cyclic GMP-selective phosphodiesterase type 5 (PDE5) has been traditionally thought to play a little role in cardiac myocytes, yet recent studies using selective inhibitors such as sildenafil suggest it can potently modulate acute and chronic cardiac stress responses. To date, evidence for myocyte PDE5 expression and regulation has relied on small-molecule inhibitors and anti-sera, leaving open concerns regarding non-specific immune-reactivity, and off-target drug effects. To directly address both issues, we engineered a robust PDE5-gene silencing shRNA (inserted into miRNA-155 cassette) and DsRed-PDE5 fusion protein, both coupled to a CMV promoter and incorporated into adenoviral vectors. PDE5 mRNA and protein knock-down eliminated anti-sera positivity on immunoblots and fluorescent immuno-histochemistry in neonatal and adult cardiomyocytes, and suppressed PDE5 enzyme activity. Stimulation of myocyte hypertrophy by phenylephrine was blunted by PDE5 gene silencing in a protein kinase G dependent manner, and this effect was similar to that from sildenafil with no additive response by both combined. DsRed-PDE5 fusion protein expression showed normal z-band localization in adult myocytes but was diffused in eNOS(-/-) myocytes; echoing reported findings with anti-sera. PDE5 overexpression increased enzyme activity and amplified natriuretic peptide gene expression from phenylephrine stimulation. These data confirm PDE5 expression, activity, and targeted inhibition by sildenafil in cardiomyocytes, as well as the role of this PDE in cardiomyocyte hypertrophy modulation.

Alveolar cell apoptosis is dependent on p38 MAP kinase-mediated activation of xanthine oxidoreductase in ventilator-induced lung injury.

Signaling via p38 MAP kinase has been implicated in the mechanotransduction associated with mechanical stress and ventilator-induced lung injury (VILI). However, the critical downstream mediators of alveolar injury remain incompletely defined. We provide evidence that high-tidal volume mechanical ventilation (HVt MV) rapidly activates caspases within the lung, resulting in increased alveolar cell apoptosis. Antagonism of MV-induced p38 MAP kinase activity with SB-203580 suppresses both MV-induced caspase activity and alveolar apoptosis, placing p38 MAP kinase upstream of MV-induced caspase activation and programmed cell death. The reactive oxygen species (ROS)-producing enzyme xanthine oxidoreductase (XOR) is activated in a p38 MAP kinase-dependent manner following HVt MV. Allopurinol, a XOR inhibitor, also suppresses HVt MV-induced apoptosis, implicating HVt MV-induced ROS in the induction of alveolar cell apoptosis. Finally, systemic administration of the pan-caspase inhibitor, z-VAD-fmk, but not its inactive peptidyl analog, z-FA-fmk, blocks ventilator-induced apoptosis of alveolar cells and alveolar-capillary leak, indicating that caspase-dependent cell death is necessary for VILI-associated barrier dysfunction in vivo.

Macrophage migration inhibitory factor governs endothelial cell sensitivity to LPS-induced apoptosis.

Human endothelial cells (EC) are typically resistant to the apoptotic effects of stimuli associated with lung disease. The determinants of this resistance remain incompletely understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by human pulmonary artery EC (HPAEC). Its expression increases in response to various death-inducing stimuli, including lipopolysaccharide (LPS). We show here that silencing MIF expression by RNA interference (MIF siRNA) dramatically reduces MIF mRNA expression and the LPS-induced increase in MIF protein levels, thereby sensitizing HPAECs to LPS-induced cell death. Addition of recombinant human MIF (rhMIF) protein prevents the death-sensitizing effect of MIF siRNA. A common mediator of apoptosis resistance in ECs is the death effector domain (DED)-containing protein, FLIP (FLICE-like inhibitory protein). We show that LPS induces a transcription-independent increase in the short isoform of FLIP (FLIP(s)). This increase is blocked by MIF siRNA but restored with the addition of recombinant MIF protein (rHMIF). While FLIP(s) siRNA also sensitizes HPAECs to LPS-induced death, the addition of rhMIF does not affect this sensitization, placing MIF upstream of FLIP(s) in preventing HPAEC death. These studies demonstrate that MIF is an endogenous pro-survival factor in HPAECs and identify a novel mechanism for its role in apoptosis resistance through the regulation of FLIP(s). These results show that MIF can protect vascular endothelial cells from inflammation-associated cell damage.

The pro-angiogenic cytokine pleiotrophin potentiates cardiomyocyte apoptosis through inhibition of endogenous AKT/PKB activity.

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3beta reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.