William Bailey

Group X secretory phospholipase A2 augments angiotensin II-induced inflammatory responses and abdominal aortic aneurysm formation in apoE-deficient mice

OBJECTIVE Abdominal aortic aneurysm (AAA) is a complex vascular disease characterized by matrix degradation and inflammation and is a major cause of mortality in older men. Specific interventions that prevent AAA progression remain to be identified. In this study, we tested the hypothesis that Group X secretory phospholipase A(2) (GX sPLA(2)), an enzyme implicated in inflammatory processes, mediates AAA. METHODS AND RESULTS GX sPLA(2) was detected by immunostaining in human aneurysmal tissue and in angiotensin II (Ang II)-induced AAAs in apolipoprotein E-deficient (apoE(-/-)) mice. GX sPLA(2) mRNA was increased significantly (11-fold) in abdominal aortas of apoE(-/-) mice in response to Ang II infusion. To define the role of GX sPLA(2) in experimental AAAs, apoE(-/-) and apoE(-/-) x GX sPLA(2)(-/-) (GX DKO) mice were infused with Ang II for either 10 (n=7) or 28 (n=24-26) days. Deficiency of GX sPLA(2) significantly reduced the incidence and severity of AAAs, as assessed by ultrasound measurements in vivo of aortic lumens and by computer-assisted morphometric analyses ex vivo of external diameter. Results from gene expression profiling indicated that the expression of specific matrix metalloproteinases and inflammatory mediators was blunted in aortas from GX DKO mice compared to apoE(-/-) mice after 10-day Ang II infusion. Ang II induction of cyclooxygenase-2, interleukin-6, matrix metalloproteinase (MMP)-2, MMP-13 and MMP-14 was reduced significantly in GX DKO mice compared to apoE(-/-) mice. CONCLUSION GX sPLA(2) promotes Ang II-induced pathological responses leading to AAA formation.

Group X Secretory Phospholipase A2 Negatively Regulates ABCA1 and ABCG1 Expression and Cholesterol Efflux in Macrophages

Objective—GX sPLA2 potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids; it has been implicated in inflammatory diseases, including atherosclerosis. To identify a novel role for group X (GX) secretory phospholipase A2 (sPLA2) in modulating ATP binding casette transporter A1 (ABCA1) and ATP binding casette transporter G1 (ABCG1) expression and, therefore, macrophage cholesterol efflux. Methods and Results—The overexpression or exogenous addition of GX sPLA2 significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA2 deficiency in mouse peritoneal macrophages was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA2-overexpressing J774 cells and increased efflux in GX sPLA2-deficient mouse peritoneal macrophages. Gene regulation was dependent on GX sPLA2 catalytic activity, mimicked by arachidonic acid and abrogated when liver X receptor (LXR)&agr;/&bgr; expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA2 suppresses the ability of LXR to transactivate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand-binding domain. Conclusion—GX sPLA2 modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA2 may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating the genes critical for cellular cholesterol efflux.

Age decreases macrophage IL-10 expression: Implications for functional recovery and tissue repair in spinal cord injury

Macrophages with different activation states are present after spinal cord injury (SCI). M1 macrophages purportedly promote secondary injury processes while M2 cells support axon growth. The average age at the time of SCI has increased in recent decades, however, little is known about how different physiological factors contribute to macrophage activation states after SCI. Here we investigate the effect of age on IL-10, a key indicator of M2 macrophage activation. Following mild-moderate SCI in 4 and 14 month old (MO) mice we detected significantly reduced IL-10 expression with age in the injured spinal cord. Specifically, CD86/IL-10 positive macrophages, also known as M2b or regulatory macrophages, were reduced in 14 vs. 4 MO SCI animals. This age-dependent shift in macrophage phenotype was associated with impaired functional recovery and enhanced tissue damage in 14-month-old SCI mice. In vitro, M2b macrophages release anti-inflammatory cytokines without causing neurotoxicity, suggesting that imbalances in the M2b response in 14-month-old mice may be contributing to secondary injury processes. Our data indicate that age is an important factor that regulates SCI inflammation and recovery even to mild-moderate injury. Further, alterations in macrophage activation states may contribute to recovery and we have identified the M2b phenotype as a potential target for therapeutic intervention.

Group X secretory phospholipase A2 negatively regulates adipogenesis in murine models

Studies in vitro indicate that group X secretory phospholipase A2 (GX sPLA2) potently releases arachidonic acid (AA) and lysophosphatidylcholine from mammalian cell membranes. To define the function of GX sPLA2 in vivo, our laboratory recently generated C57BL/6 mice with targeted deletion of GX sPLA2 (GX−/− mice). When fed a normal rodent diet, GX−/− mice gained significantly more weight and had increased adiposity compared to GX+/+ mice, which was not attributable to alterations in food consumption or energy expenditure. When treated with adipogenic stimuli ex vivo, stromal vascular cells isolated from adipose tissue of GX−/− mice accumulated significantly more (20%) triglyceride compared to cells from GX+/+ mice. Conversely, overexpression of GX sPLA2, but not catalytically inactive GX sPLA2, resulted in a significant 50% reduction in triglyceride accumulation in OP9 adipocytes. The induction of genes encoding adipogenic proteins (PPARγ, SREBP‐1c, SCD1, and FAS) was also significantly blunted by 50–80% in OP9 cells overexpressing GX sPLA2. Activation of the liver X receptor (LXR), a nuclear receptor known to up‐regulate adipogenic gene expression, was suppressed in 3T3‐L1 and OP9 cells when GX sPLA2 was overexpressed. Thus, hydrolytic products generated by GX sPLA2 negatively regulate adipogenesis, possibly by suppressing LXR activation.—Li, X., Shridas, P., Forrest, K., Bailey, W., Webb, N. R. Group X secretory phospholipase A2 negatively regulates adipogenesis in murine models. FASEB J. 24, 4313–4324 (2010). www.fasebj.org

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A2 (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A2 (sPLA2), but not a catalytically inactive mutant form of GX sPLA2, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA2 inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA2-overexpressing Y1 cells, ruling out a role for this sPLA2 receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA2. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA2 antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA2 is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

Group X Secretory Phospholipase A2 Enhances TLR4 Signaling in Macrophages

Secretory phospholipase A2s (sPLA2) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA2 is recognized as the most potent mammalian sPLA2 in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA2 suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014–2021). In this study, we provide evidence that GX sPLA2 modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA2 resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA2 catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA2 was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA2-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA2-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA2 amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.

Predictive screening of M1 and M2 macrophages reveals the immunomodulatory effectiveness of post spinal cord injury azithromycin treatment.

Spinal cord injury (SCI) triggers a heterogeneous macrophage response that when experimentally polarized toward alternative forms of activation (M2 macrophages) promotes tissue and functional recovery. There are limited pharmacological therapies that can drive this reparative inflammatory state. In the current study, we used in vitro systems to comprehensively defined markers of macrophages with known pathological (M1) and reparative (M2) properties in SCI. We then used these markers to objectively define the macrophage activation states after SCI in response to delayed azithromycin treatment. Mice were subjected to moderate-severe thoracic contusion SCI. Azithromycin or vehicle was administered beginning 30 minutes post-SCI and then daily for 3 or 7 days post injury (dpi). We detected a dose-dependent polarization toward purportedly protective M2 macrophages with daily AZM treatment. Specifically, AZM doses of 10, 40, or 160 mg/kg decreased M1 macrophage gene expression at 3 dpi while the lowest (10 mg/kg) and highest (160 mg/kg) doses increased M2 macrophage gene expression at 7 dpi. Azithromycin has documented immunomodulatory properties and is commonly prescribed to treat infections in SCI individuals. This work demonstrates the utility of objective, comprehensive macrophage gene profiling for evaluating immunomodulatory SCI therapies and highlights azithromycin as a promising agent for SCI treatment.

Deficiency of Endogenous Acute-Phase Serum Amyloid A Protects apoE−/− Mice From Angiotensin II–Induced Abdominal Aortic Aneurysm Formation

Objective— Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. Approach and Results— Plasma SAA increased ≈60-fold in apoE−/− mice 24 hours after intraperitoneal Ang II injection (100 &mgr;g/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE−/− mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE−/− mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II–infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. Conclusions— Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.

Age increases reactive oxygen species production in macrophages and potentiates oxidative damage after spinal cord injury.

Age potentiates neurodegeneration and impairs recovery from spinal cord injury (SCI). Previously, we observed that age alters the balance of destructive (M1) and protective (M2) macrophages; however, the age-related pathophysiology in SCI is poorly understood. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) contributes to reactive oxygen species (ROS)-mediated damage and macrophage activation in neurotrauma. Further, NOX and ROS increase with central nervous system age. Here, we found significantly higher ROS generation in 14 versus 4-month-old (MO) mice after contusion SCI. Notably, NOX2 increased in 14 MO ROS-producing macrophages suggesting that macrophages and NOX contribute to SCI oxidative stress. Indicators of lipid peroxidation, a downstream cytotoxic effect of ROS accumulation, were significantly higher in 14 versus 4 MO SCI mice. We also detected a higher percentage of ROS-producing M2 (Arginase-1-positive) macrophages in 14 versus 4 MO mice, a previously unreported SCI phenotype, and increased M1 (CD16/32-positive) macrophages with age. Thus, NOX and ROS are age-related mediators of SCI pathophysiology and normally protective M2 macrophages may potentiate secondary injury through ROS generation in the aged injured spinal cord.

Group V secretory phospholipase A2 enhances the progression of angiotensin II-induced abdominal aortic aneurysms but confers protection against angiotensin II-induced cardiac fibrosis in apoE-deficient mice.

Abdominal aortic aneurysms (AAAs) and heart failure are complex life-threatening diseases whose etiology is not completely understood. In this study, we investigated whether deficiency of group V secretory phospholipase A(2) (GV sPLA(2)) protects from experimental AAA. The impact of GV sPLA(2) deficiency on angiotensin (Ang) II-induced cardiac fibrosis was also investigated. Apolipoprotein E (apoE)(-/-) mice and apoE(-/-) mice lacking GV sPLA(2) (GV DKO) were infused with 1000 ng/kg per minute Ang II for up to 28 days. Increases in systolic blood pressure, plasma aldosterone level, and urinary and heart prostanoids were similar in apoE(-/-) and GV DKO mice after Ang II infusion. The incidence of aortic rupture in Ang II-infused GV DKO mice (10%) was significantly reduced compared with apoE(-/-) mice (29.4%). Although the incidence of AAA in GV DKO mice (81.3%) and apoE(-/-) mice (100%) was similar, the mean percentage increase in maximal luminal diameter of abdominal aortas was significantly smaller in GV DKO mice (68.5% ± 7.7%) compared with apoE(-/-) mice (92.6% ± 8.3%). Deficiency of GV sPLA(2) resulted in increased Ang II-induced cardiac fibrosis that was most pronounced in perivascular regions. Perivascular collagen, visualized by picrosirius red staining, was associated with increased TUNEL staining and increased immunopositivity for macrophages and myofibroblasts and nicotinamide adenine dinucleotide phosphate oxidase (NOX)-2 and NOX-4, respectively. Our findings indicate that GV sPLA(2) modulates pathological responses to Ang II, with different outcomes for AAA and cardiac fibrosis.