Katy Maguin-Gaté

Physical training and hypertension have opposite effects on endothelial brain-derived neurotrophic factor expression

AIMS Changes in circulating brain-derived neurotrophic factor (BDNF) levels were reported in patients with or at risk for cardiovascular diseases associated with endothelial dysfunction, suggesting a link between BDNF and endothelial functionality. However, little is known on cardiovascular BDNF. Our aim was to investigate levels/localization, function, and relevance of cardiovascular BDNF. METHODS AND RESULTS BDNF levels (western blotting) and localization (immunostaining) were assessed in the heart and aorta from rats with impaired (spontaneously hypertensive rats [SHR]), normal (Wistar Kyoto rats [WKY]), and improved (SHR and WKY subjected to physical training) endothelial function. BDNF levels were also measured in cultured endothelial cells (CECs) subjected to low and high shear stress. The cardiovascular effects of BDNF were investigated in isolated aortic rings and hearts. The results showed high BDNF levels in the heart and aorta, the expression being prominent in endothelial cells as compared with other cell types. Exogenous BDNF vasodilated aortic rings but changed neither coronary flow nor cardiac contractility. Hypertension was associated with decreased expression of BDNF in the endothelium, whereas physical training led to endothelial BDNF up-regulation not only in WKY but also in SHR. Exposure of CECs to high shear stress stimulated BDNF production and secretion. CONCLUSION Cardiovascular BDNF is mainly localized within endothelial cells in which its expression is dependent on endothelial function. These results open new perspectives on the role of endothelial BDNF in cardiovascular health.

Mechanisms of endothelial dysfunction in rheumatoid arthritis: lessons from animal studies

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by articular and extra-articular manifestations involving cardiovascular diseases (CVDs), which account for 30% to 50% of all deaths. In patients with RA, atherosclerosis lesions occur earlier and have a more rapid evolution than in the general population. Beyond mortality, the impact of CVD on quality of life, combined with the associated increase in health-care costs, renders CVD in RA a major public health problem. Recent studies showed that patients with RA are characterized by the presence of endothelial dysfunction (ED), which is recognized as a key event in the development of atherosclerosis. By definition, ED is a functional and reversible alteration of endothelial cells, leading to a shift of the actions of the endothelium toward reduced vasodilation, proinflammatory state and proliferative and prothrombotic properties. Although the improvement of endothelial function is becoming an important element of the global management of patients with RA, the mechanistic determinants of ED in RA are still poorly understood. Animal models of RA provide the unique opportunity to unravel the pathophysiological features of ED in RA. The present review summarizes the available data on mechanisms underlying ED in animal models of RA and proposes attractive prospects in order to discover novel therapeutic strategies of RA-associated ED.

Endothelial Dysfunction in Rheumatoid Arthritis: Mechanistic Insights and Correlation with Circulating Markers of Systemic Inflammation

Objectives To determine mechanisms involved in endothelial dysfunction (ED) during the course of arthritis and to investigate the link between cytokines, chemokines and osteoprotegerin. Approach and Results Experiments were conducted on aortic rings at day 4 (preclinical), day 11 (onset of disease), day 33 (acute disease) and day 90 (chronic disease) after adjuvant-induced arthritis (AIA) in Lewis rats. At day 4, the unique vascular abnormality was a reduced norepinephrine-induced constriction. At day 11, endothelial function assessed by the relaxation to acetylcholine was normal despite increased cyclo-oxygenase-2 activity (COX-2) and overproduction of superoxide anions that was compensated by increased nitric oxide synthase (NOS) activity. At day 33, ED apparition coincides with the normalization of NOS activity. At day 90, ED was only observed in rats with a persisting imbalance between endothelial NOS and COX-2 pathways and higher plasma levels of IL-1β and TNFα. Plasma levels of IL-1β, TNFα and MIP-1α negatively correlated with Ach-induced relaxation throughout the course of AIA. Conclusions Our data identified increased endothelial NOS activity as an important compensatory response that opposes the ED in the early arthritis. Thereafter, a cross-talk between endothelial COX-2/NOS pathways appears as an important element for the occurrence of ED. Our results encourage determining the clinical value of IL-1β, TNFα and MIP-1α as biomarkers of ED in RA.

Etanercept improves endothelial function via pleiotropic effects in rat adjuvant-induced arthritis

OBJECTIVES To determine the effect of etanercept on endothelial dysfunction and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS At the first signs of arthritis, etanercept (10 mg/kg/3 days, s.c.) or saline was administered for 3 weeks in AIA rats. Body weights and arthritis scores were monitored daily. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclo-oxygenase (COX-2), arginase, endothelium-derived hyperpolarizing factor and superoxide anions (O2 (-)°) production. Aortic expression of endothelial nitic oxide synthase (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22(phox) and p47(phox) was evaluated by western blotting analysis. Blood pressure, heart rate and blood levels of triglycerides, cholesterol and glucose were measured. RESULTS Etanercept significantly reduced arthritis score (P < 0.001). It improved Ach-induced relaxation (P < 0.05) as a result of increased NOS activity, decreased COX-2/arginase activities and decreased O2 (-)° production. These functional effects relied on increased eNOS expression and phosphorylation, and decreased COX-2, arginase-2 and p22(phox) expressions. No correlation was found between arthritis score and Ach-induced relaxation. The treatment did not change triglycerides, cholesterol and glucose levels, but significantly increased systolic blood pressure and heart rate (P < 0.05). CONCLUSION Our data demonstrated that efficient dosage of etanercept on inflammatory symptoms improved endothelial function in AIA. This beneficial effect on endothelial function is disconnected from its impact on CV risk factors and relates to pleiotropic effects of etanercept on endothelial pathways. These results suggest that etanercept could be a good choice for patients with rheumatoid arthritis at high risk of CV events.

Microvascular Abnormalities in Adjuvant‐Induced Arthritis: Relationship to Macrovascular Endothelial Function and Markers of Endothelial Activation

To determine the time course of microvascular abnormalities and the link with macrovascular endothelial function and circulating markers of endothelial activation in adjuvant‐induced arthritis (AIA) in rats.

Glucocorticoids Improve Endothelial Function in Rheumatoid Arthritis: A Study in Rats with Adjuvant‐induced Arthritis

To determine the effect of glucocorticoids (GCs) on endothelial dysfunction (ED) and on traditional cardiovascular (CV) risk factors in the adjuvant‐induced arthritis (AIA) rat model. At the first signs of AIA, a high dose (HD) [10 mg/kg/day, intraperitoneally (i.p.), GC‐HD] or low dose (LD) (1 mg/kg/day, i.p., GC‐LD) of prednisolone was administered for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclooxygenase 2 (COX‐2), arginase, endothelium derived hyperpolarizing factor (EDHF) and superoxide anions ( O2– °) production. Aortic expression of endothelial NOS (eNOS), Ser1177‐phospho‐eNOS, COX‐2, arginase‐2, p22phox and p47phox was evaluated by Western blotting analysis. Arthritis scores, blood pressure, heart rate and blood levels of cytokines, triglycerides, cholesterol and glucose were measured. GC‐HD but not GC‐LD reduced arthritis score significantly and improved Ach‐induced relaxation (P < 0·05). The positive effect of GC‐HD resulted from increased NOS activity and EDHF production and decreased COX‐2/arginase activities and O2– ° production. These functional effects relied upon increased phospho‐eNOS expression and decreased COX‐2, arginase‐2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression. Despite the lack of effect of GC‐LD on ED, it increased NOS and EDHF and down‐regulated O2– ° pathways but did not change arginase and COX‐2 pathways. GC‐HD increased triglycerides levels and blood pressure significantly (P < 0·05). Both doses of GCs decreased to the same extent as plasma interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α levels (P < 0·05). Our data demonstrated that subchronic treatment with prednisolone improved endothelial function in AIA via pleiotropic effects on endothelial pathways. These effects occurred independently of the deleterious cardiometabolic effects and the impact of prednisolone on systemic inflammation.

Local cryotherapy improves adjuvant-induced arthritis through down-regulation of IL-6 / IL-17 pathway but independently of TNFα

Objectives Local cryotherapy is widely and empirically used in the adjuvant setting in rheumatoid arthritis treatment, however its own therapeutic and anti-inflammatory effects are poorly characterized. We aimed to evaluate the effects of local cryotherapy on local and systemic inflammation in Adjuvant-induced arthritis, a murine model of rheumatoid arthritis. Methods The effects of mild hypothermia (30°C for 2 hours) on cytokine protein levels (Multiplex/ELISA) were evaluated in vitro in cultured rat adjuvant-induced arthritis patellae. In vivo, local cryotherapy was applied twice a day for 14 days in arthritic rats (ice: n = 10, cold gas: n = 9, non-treated: n = 10). At day 24 after the induction of arthritis, cytokine expression levels were measured in grinded hind paws (Q-RT-PCR) and in the plasma (Multiplex/ELISA). Results In vitro, punctual mild hypothermia down-regulated IL-6 protein expression. In vivo, ice showed a better efficacy profile on the arthritis score and joint swelling and was better tolerated, while cold gas induced a biphasic response profile with initial, transient arthritis worsening. Local cryotherapy also exerted local and systemic anti-inflammatory effects, both at the gene and the protein levels: IL-6, IL-17A and IL-1β gene expression levels were significantly down-regulated in hind paws. Both techniques decreased plasma IL-17A while ice decreased plasma IL-6 protein levels. By contrast, we observed no effect on local/systemic TNF-α pathway. Conclusions We demonstrated for the first time that sub-chronically applied local cryotherapy (ice and cold gas) is an effective and well-tolerated treatment in adjuvant-induced arthritis. Furthermore, we provided novel insights into the cytokine pathways involved in Local cryotherapy’s local and systemic anti-inflammatory effects, which were mainly IL-6/IL-17A-driven and TNF-α independent in this model.

AB0089 Non-Steroidal Anti-Inflammatory Drugs and Endothelial Function in Ra: Harmful or Beneficial? A Study in Rat Adjuvant-Induced Arthritis

Background Rheumatoid Arthritis (RA) is associated with increased cardiovascular risk (CVR) explained in part by accelerated atherosclerosis as a consequence of endothelial dysfunction. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely prescribed in RA patients in case of acute pain. Surprisingly, despite the commonly-held belief that NSAIDs worsen CVR, data regarding their effect on endothelial function in RA are lacking. Objectives The present study investigated the effect of naproxen (a non-selective COX inhibitor), diclofenac (a preferential COX-2 inhibitor) and celecoxib (a selective COX-2 inhibitor) on vascular function in arthritic rats and identified the underlying mechanisms. Methods Adjuvant-induced arthritis (AIA) was induced in 6 weeks-old male Lewis rats by injection of Mycobacterium butyricum in adjuvant at the basis of the tail. At the onset of arthritis, rats were daily treated (i.p.) with naproxen (10 mg/kg/d), diclofenac (5mg/kg twice a day), celecoxib (3 mg/kg/d) or saline (Vehicle) for 21 days. Arthritis scores were daily monitored. At the end of treatment, thoracic aortas were harvested to measure the relaxation to acetylcholine (Ach) on pre-constricted aortic rings in the presence or not of inhibitor of nitric oxide (NO) synthase (L-NAME), arginase (nor-NOHA), EDHF (Apamin/Charybdotoxin), or a superoxide dismutase analog (Tempol). The effects of a NO donor (sodium nitroprussiate, SNP) and norepinephrine (NE) were studied on endothelium-denuded aortic rings. Results All NSAIDs significantly reduced to the same extent arthritis score in AIA (p<0.05). By contrast, differences exist between NSAIDs regarding endothelial function. Both naproxen and diclofenac significantly (p<0.05) improved Ach-induced relaxation whereas celecoxib did not change the response to Ach. These differences on endothelial function were not explained by differences in the response of vascular smooth muscle to NE or SNP which remained unchanged after the 3 treatments. Of interest, the positive effects of naproxen and diclofenac likely relied on different mechanisms. Whilst naproxen tends to improve NOS activity and decrease superoxide anion production, diclofenac decreased arginase activity, superoxide anion production and improved EDHF production. Conclusions Our study demonstrates for the first time that naproxen and diclofenac have positive effects on endothelial function in case of arthritis whereas celecoxib had no effect, despite a similar reduction of inflammatory symptoms. From a clinical perspective, these data highlight the heterogeneity of effects of NSAIDs as regards CVR in RA and encourage designing clinical trials to confirm our experimental data and help guiding the better choice of NSAIDs for the global management of RA patients. Disclosure of Interest None declared

AB0119 High Dose Glucocorticoids Are Not Deleterious for Endothelial Function in Rheumatoid Arthritis: Mechanistic Insights in Adjuvant-Induced Arthritis

Background Rheumatoid Arthritis (RA) is associated to an increase in cardiovascular risk (CVR) explained in part by accelerated atherosclerosis as a consequence of endothelial dysfunction. Glucocorticoids (GCs) are widely prescribed in RA patients. Surprisingly, despite the commonly held belief that glucocorticoids worsen the CVR, data concerning their impact on endothelial function in RA is lacking. Objectives The present study investigated the effect of a high dose of prednisolone on vascular function in arthritic rats and identified the underlying mechanisms. Methods Adjuvant-induced arthritis (AIA) was induced in 6-week-old male Lewis rats by injection of Mycobacterium butyricum in adjuvant at the basis of the tail. At the onset of arthritis, rats were daily treated (i.p.) with prednisolone (10 mg /kg, AIA-GC) or saline (Vehicle) for 21 days. Arthritis score and tarsus diameter were daily monitored. At the end of treatment, thoracic aortas were harvested to measure the relaxation of pre-constricted aortic rings to acetylcholine in the presence or not of inhibitor of nitric oxide synthase (NOS) (L-NAME), arginase (nor-NOHA), COX-2 (NS-398), EDHF (Apamin/Charybdotoxin) or a superoxide dismutase analog (Tempol). The effect of a NO donor (sodium nitroprussiate, SNP) and norepineprhine (NE) was studied on endothelium-denuded aortic rings. Aortic expression of endothelial NOS (eNOS) and phospho-eNOS (Ser-1177) was evaluated by western blotting. Blood pressure, glycaemia, triglyceride and total cholesterol levels and radiological score of hind paws were also assessed. Results Consistent with the well-known effects of a high dose of GC, AIA-GC exhibited significantly higher blood pressure (p<0.05) and triglyceride levels (p<0.05) compared to AIA-vehicle (p<0.05). Glycemia was unchanged and total cholesterol levels were lower (p<0.01) in treated compared to untreated AIA. Arthritic scores, paw diameters and radiological damage were dramatically reduced by prednisolone (p<0.001). In endothelium-denuded rings, vascular reactivity to SNP or to NE was unaffected by GC. By contrast, GC significantly improved endothelial function as attested by enhanced Ach-induced relaxation compared to AIA-vehicle. This beneficial effect relied on increased phosphorylation of NOS and a subsequent enhancement of NO synthase activity, increased EDHF production and decreased superoxide anions production and activity of arginase and COX-2. Conclusions Our study demonstrated that a high dose of prednisolone improved endothelial function in case of RA, independently of its deleterious cardio-metabolic effects. From a clinical perspective, these results raise the question of the use of high doses of GC during a short period to overcome CV risk along with a rapid disease control. Whether this beneficial vascular effect of GC depends on the reduction of disease activity or not deserves further investigations. Disclosure of Interest None declared

AB0138 Time-Course of Microvascular versus Macrovascular Endothelial Dysfunction in Rat Adjuvant-Induced Arthritis

Background Rheumatoid arthritis (RA) is associated with increased risk for cardiovascular disease (CVD). Whereas ample evidence demonstrated the presence of endothelial dysfunction (ED) in RA, it is still unclear how early this abnormality develops. Moreover most of studies have been performed on the macrovasculature but little is known about ED in the microvasculature. Objectives The aim of this study is to investigate the time-course of microvascular and macrovascular impairment in adjuvant-induced arthritis (AIA) in rats. Methods AIA was induced by an intradermal injection of Mycobacterium butyricum in the tail of male Lewis rats (Day 0). Arthritis severity was evaluated by clinical and radiological analysis at 4 stages of AIA: day 4 (preclinical stage), day 11 (very early arthritis), day 33 (severe disease), day 90 (chronic disease). At each stage, functional and mechanical properties of third-order mesenteric arteries were determined by assessing flow-mediated dilatation, myogenic tone and stress-strain relationship. Macrovascular function was studied on isolated aortic rings in which the contractile response to norepinephrine (NE) and the vasorelaxant response to Acetylcholine (Ach) and to sodium nitroprussiate (SNP) were evaluated. Results Macrovascular ED became evident at the severe disease. At the chronic phase of AIA the presence of ED was positively correlated to the radiological score. The response of aortic rings to SNP was normal whatever the stage of AIA. By contrast, NE-induced contractility was significantly reduced in AIA at day 4, and recovered the normal values thereafter. In microvascular bed, ED appears at the early arthritis stage. At the severe disease, microvascular ED is still present and is associated with reduced arterial stiffness. At day 90, one group of microvessels from AIA exhibited ED whereas another group did not, but ED was not correlated to the radiological score. Myogenic tone was unchanged during the course of arthritis. Conclusions Our results demonstrated that micro and macrovascular beds are differentially affected during the course of RA. Microvascular ED is a very early event in the course of arthritis. It precedes the occurrence of macrovascular ED and microvascular stiffness. Our data suggest that the measurement of microvascular function would be relevant for the early diagnosis of CV risk in RA patients. They also suggest that mechanisms underlying macro- and microvascular dysfunction are different. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1687