Damien Calay

PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis.

Aims Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKCε) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKCε. Methods and results Expression of constitutively active PKCε in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKCε-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKCε and induced HO-1 via a PKCε-dependent pathway. PKCε activation significantly attenuated TNFα-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1(Ser133) phosphorylation as a PKCε signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKCε-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKCε target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKCε-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKCε-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2. Conclusions PKCε activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKCε in the maintenance of endothelial homeostasis and resistance to injury.

Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation

Aims Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes. Methods/results In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1u2005mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKαThr172, and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)Ser133 phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKαThr172 phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. Conclusions These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

Protein kinase Cε activity induces anti-inflammatory and anti-apoptotic genes via an ERK1/2- and NF-κB-dependent pathway to enhance vascular protection.

Vascular endothelial injury predisposes to endothelial dysfunction and atherogenesis. We have investigated the hypothesis that PKCε (protein kinase Cε) is an important upstream regulator of cytoprotective pathways in vascular ECs (endothelial cells). Depletion of PKCε in human ECs reduced expression of the cytoprotective genes A1, A20 and Bcl-2. Conversely, constitutively active PKCε expressed in human ECs increased mRNA and protein levels of these cytoprotective genes, with up-regulation dependent upon ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. Furthermore, inhibition of NF-κB (nuclear factor κB) by the pharmacological antagonist BAY 11-7085 or an IκB (inhibitor of NF-κB) SuperRepressor prevented cytoprotective gene induction. Activation of PKCε enhanced p65 NF-κB DNA binding and elevated NF-κB transcriptional activity. Importantly, although NF-κB activation by PKCε induced cytoprotective genes, it did not up-regulate pro-inflammatory NF-κB targets [E-selectin, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1)]. Indeed, PKCε exhibited cytoprotective and anti-inflammatory actions, including inhibition of TNFα (tumour necrosis factor α)-induced JNK (c-Jun N-terminal kinase) phosphorylation and ICAM-1 up-regulation, a response attenuated by depletion of A20. Thus we conclude that PKCε plays an essential role in endothelial homoeostasis, acting as an upstream co-ordinator of gene expression through activation of ERK1/2, inhibition of JNK and diversion of the NF-κB pathway to cytoprotective gene induction, and propose that PKCε represents a novel therapeutic target for endothelial dysfunction.

Synergistic Therapeutic Vascular Cytoprotection against Complement-Mediated Injury Induced via a PKCα-, AMPK-, and CREB-Dependent Pathway

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1−/− mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis.

Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis.

Celecoxib exerts protective effects in the vascular endothelium via COX-2-independent activation of AMPK-CREB-Nrf2 signalling

Although concern remains about the athero-thrombotic risk posed by cyclo-oxygenase (COX)-2-selective inhibitors, recent data implicates rofecoxib, while celecoxib appears equivalent to NSAIDs naproxen and ibuprofen. We investigated the hypothesis that celecoxib activates AMP kinase (AMPK) signalling to enhance vascular endothelial protection. In human arterial and venous endothelial cells (EC), and in contrast to ibuprofen and naproxen, celecoxib induced the protective protein heme oxygenase-1 (HO-1). Celecoxib derivative 2,5-dimethyl-celecoxib (DMC) which lacks COX-2 inhibition also upregulated HO-1, implicating a COX-2-independent mechanism. Celecoxib activated AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation. Importantly, these responses were not reproduced by ibuprofen or naproxen, while AMPKα silencing abrogated celecoxib-mediated CREB and Nrf2 activation. Moreover, celecoxib induced H-ferritin via the same pathway, and increased HO-1 and H-ferritin in the aortic endothelium of mice fed celecoxib (1000u2009ppm) or control chow. Functionally, celecoxib inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by activating AMPK. This attenuated VCAM-1 upregulation via induction of HO-1, a response reproduced by DMC but not ibuprofen or naproxen. Similarly, celecoxib prevented IL-1β-mediated induction of IL-6. Celecoxib enhances vascular protection via AMPK-CREB-Nrf2 signalling, a mechanism which may mitigate cardiovascular risk in patients prescribed celecoxib. Understanding NSAID heterogeneity and COX-2-independent signalling will ultimately lead to safer anti-inflammatory drugs.

THU0053 Selective activation of an AMPK-CREB-NRF2-dependent pathway by celecoxib induces vasculoprotective genes and mitigates against cardiovascular risk

Background Although concern remains about the athero-thrombotic risk posed by COX-2-selective inhibitors (COXIBs), the recent PRECISION trial demonstrated non-inferiority of moderate dose celecoxib when compared to naproxen and ibuprofen with respect to cardiovascular safety, with fewer actual CV events recorded for celecoxib. Moreover, celecoxib proved significantly safer than either comparator in regard to gastrointestinal events1. Given the markedly different cardiovascular risk associated with celecoxib and rofecoxib, we investigated the hypothesis that, in addition to cyclo-oxygenase inhibition, celecoxib specifically activates COX-2-independent AMP kinase (AMPK) signalling to exert protective effects in the vascular endothelium. Objectives To investigate COX-2-independent vasculoprotective signalling pathways activated by celecoxib in human endothelium. Methods In vitro studies of celecoxib, rofecoxib, ibuprofen and naproxen were performed on human umbilical vein and human aortic endothelial cells (HUVEC and HAEC). Inhibition of signalling pathways was achieved using siRNA. The vascular effects of celecoxib in vivo were studied in C57Bl/6 mice fed celecoxib (1000 ppm) or control chow (48 hrs). Aortic tissue was snap-frozen and sections studied by immunofluorescence confocal microscopy. Results At therapeutically relevant concentrations celecoxib (1–10μ M) induced the vasculoprotective protein heme oxygenase-1 (HO-1) in HUVEC and HAEC (EC) (p<0.01). In contrast, rofecoxib and the commonly used non-selective NSAIDs ibuprofen and naproxen failed to induce HO-1. Celecoxib derivative 2,5-dimethyl-celecoxib (DMC), which lacks COX-2 inhibition, also upregulated HO-1, implicating a COX-2-independent mechanism. Immunoblotting demonstrated that celecoxib and DMC induce AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation (p<0.05). These responses were not seen with ibuprofen or naproxen, while siRNA depletion of AMPKα abrogated celecoxib-mediated CREB and Nrf2 activation (p<0.05). Acting via the same pathway, celecoxib induced additional cytoprotective genes including H-ferritin. In vivo, celecoxib similarly increased HO-1 and H-ferritin in murine aortic endothelium when compared to control-fed mice (p<0.05). Functionally, celecoxib treatment inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by increasing AMPK activity. This attenuated VCAM-1 upregulation via induction of HO-1, as revealed by HO-1 siRNA (p<0.05). Similarly, celecoxib prevented the IL-1-mediated increase of IL-6 mRNA (p<0.01). These responses were not seen with ibuprofen or naproxen. Conclusions Celecoxib induces anti-inflammatory, anti-oxidant proteins HO-1 and H-ferritin in human vascular endothelium via a novel AMPK-CREB-Nrf2-dependent pathway. This mechanism may contribute to the important and marked differences in cardiovascular risk between celecoxib and rofecoxib. Understanding mechanisms underlying NSAID heterogeneity may ultimately lead to the development of safer anti-inflammatory drugs. References Nissen SE et al. Cardiovascular Safety of Celecoxib, Naproxen, or Ibuprofen for Arthritis. N Engl J Med. 2016;375:2519–29. Disclosure of Interest None declared

THU0522 Methotrexate: A Novel Mechanism for Vasculoprotection in Chronic Systemic Inflammation

Background Chronic systemic inflammation is associated with endothelial dysfunction leading to accelerated atherosclerosis. Although treatment of inflammatory disease has significantly improved, there is as yet no specific strategy to protect the vasculature and retard the onset of cardiovascular disease (CVD).Clinical data suggests that long term low-dose methotrexate (MTX) reduces CV mortality in rheumatoid arthritis. However, little is known about the mechanisms underlying this. Known actions of MTX include inhibition of purine synthesis enzymes, resulting in intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide. These are activators of AMP-activated protein kinase (AMPK), a ubiquitous signalling kinase that regulates cytoprotective genes in endothelial cells (EC) including manganese superoxide dismutase (MnSOD) and heme oxygenase-1 (HO1). We hypothesised that MTX exerts beneficial protective effects on vascular endothelium via activation of AMPK which enhance its ability to withstand inflammatory-mediated injury. Objectives This study aimed to establish whether MTX can activate AMPK and induce cytoprotective gene expression in EC, explore the intermediaries involved, study the functional relevance of this pathway and whether MTX affects endothelial cell survival.Finally, we planned to treat a model of inflammatory vascular disease with MTX to establish whether it protects vascular endothelium in vivo. Methods In vitro studies of the effects of MTX were carried out on human umbilical vein EC (HUVEC), using western blotting, qRTPCR, flow cytometry and chromatin immunoprecipitation.Inhibition of signalling pathways was achieved using short interfering RNA. The vascular effects of MTX in vivo were studied in the murine (NZWxBXSB) F1 (WBF1) model of SLE vasculopathy.Animals were treated with MTX 1mg/kg weekly by intraperitoneal injection for 4 weeks.Aortic tissue was used in Western blots and in frozen sections for immunofluorescence.Myocardial sections were stained with periodic acid-Schiff and picrosirius red. Results Treatment of HUVEC with MTX 100nM for 48h phosphorylated AMPKα and induced expression of several cytoprotective genes, notably MnSOD and HO1, while siRNA depletion of AMPK attenuated these changes.MTX treatment led to AMPK-dependent phosphorylation of the transcription factor CREB, siRNA depletion of CREB1 reduced MnSOD and HO1 induction by MTX, and chromatin immunoprecipitation demonstrated binding of CREB1 to the MnSOD promoter in MTX-treated samples.The magnitude of MnSOD induction was preserved when HUVEC were pre-treated with TNFα to mimic a dysfunctional endothelium.Finally, MTX protected HUVEC against apoptosis induced by glucose deprivation. Treating WBF1 mice with MTX reduced end-organ damage and the severity of the vasculopathy.Increased aortic expression of MnSOD and phosphorylated AMPK and reduced ICAM1 expression was observed, confirming that low dose weekly MTX can activate AMPK in the vasculature in vivo, and is associated with improvement in vascular inflammation. Conclusions MTX activates an AMPK-CREB pathway in endothelium leading to enhanced expression of cytoprotective genes and protection against apoptosis in vitro and inflammatory vascular injury in vivo. This novel mechanism may explain its observed benefits in reducing CVD in chronic systemic inflammation. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4685