Association of Soluble ST2 With Functional Outcome, Perihematomal Edema, and Immune Response After Intraparenchymal Hemorrhage.

BACKGROUND AND OBJECTIVES: Perihematomal edema (PHE) contributes to poor outcome after deep intraparenchymal hemorrhage (IPH), which is characterized by neuroinflammation and an influx of peripherally derived innate immune cells. We previously identified soluble ST2 (sST2) as a candidate for immune-mediated secondary brain injury. Leveraging prospectively collected cohorts from 2 centers, we sought to determine whether sST2 was associated with functional outcome, PHE, and the immune response following IPH. METHODS: Patients with deep IPH were enrolled within 36 hours of ictus, and blood was collected for sST2 and immune cell measurement. Hematoma volume and PHE were measured on serial CT scans. Good outcome was defined as a modified Rankin Scale score of 0-3 at 90 days. Linear mixed-effects models were used to analyze the relationship between sST2 and PHE over time. Flow cytometry was used to identify shifts in immune cell populations associated with sST2. Immunohistochemistry of human brain tissue was used to identify ST2-expressing cells in the perihematomal region. RESULTS: The 55 included patients had a median admission Glasgow Coma Scale score of 14 (interquartile range [IQR] 9-15), an intracerebral hemorrhage (ICH) score of 1 (IQR 1-2), and a hematoma volume of 8.6 mL (IQR 3.4-13.8 mL). Receiver operating curve analysis found the sST2 level to be predictive of poor outcome with an area under the curve of 0.763 (95% CI 0.632-0.894) and Youden optimum cut point of 61.8 ng/mL (p < 0.001). sST2 remained an independent predictor after adjustment for ICH score (adjusted odds ratio 2.53, 95% CI 1.03-6.19, p = 0.042). Measurement of PHE found those patients with high sST2 to have greater edema volume over time (ß = 1.07, 95% CI 0.51-1.63, p < 0.001). High sST2 was associated with a shift toward an innate peripheral immune response (monocytes and natural killer cells; 68.6% ± 5.1% vs 47.5% ± 4.0%; p = 0.003). DISCUSSION: Our findings demonstrate that elevated sST2 links the peripheral innate immune response to PHE volume and outcome after IPH. This knowledge is relevant to future studies that seek to identify patients with IPH at highest risk for immune-mediated injury or limit injury through targeted interventions.

Role of Interleukin-1 Receptor-Like 1 (ST2) in Cerebrovascular Disease.

Following both ischemic and hemorrhagic stroke, innate immune cells initiate a proinflammatory response that further exacerbate tissue injury in the acute phase, but these cells also play an important reparative role thereafter. Numerous cytokines and signaling pathways have been implicated in driving the deleterious proinflammatory response, but less is known about the mediators that connect the initial vascular injury to the systemic immune response and the relationship between proinflammatory and reparative immune responses. The Interleukin-33 (IL-33) and serum stimulation-2 (ST2) axis is an interleukin signaling pathway that is a prime candidate to fulfill this role. In this review, we describe the biology of the IL-33/ST2 system, present evidence that its soluble decoy receptor, soluble ST2 (sST2), plays a key role in secondary neurologic injury after stroke, and discuss this in the context of the known role of IL-33/ST2 in other disease.

Continuous Glibenclamide Prevents Hemorrhagic Transformation in a Rodent Model of Severe Ischemia-Reperfusion.

BACKGROUND: Endovascular thrombectomy (EVT) is highly effective but may also lead to hemorrhagic transformation (HT) and edema, which may be more pronounced in severe ischemia. We sought to determine whether glibenclamide can attenuate HT and edema in a severe ischemia-reperfusion model that reflects EVT. METHODS: Using a transient middle cerebral artery occlusion (tMCAo) rodent model of stroke, we studied two rat cohorts, one without rt-PA and a second cohort treated with rt-PA. Glibenclamide or vehicle control was administered as an intravenous bolus at reperfusion, followed by continuous subcutaneous administration with an osmotic pump. RESULTS: Compared to vehicle control, glibenclamide improved neurological outcome (median 7, interquartile range [IQR 6-8] vs. control median 6 [IQR 0-6], p = 0.025), reduced stroke volume (323 ± 42 vs. 484 ± 60 mm3, p < 0.01), swelling volume (10 ± 4 vs. 28 ± 7%, p < 0.01) and water content (84 ± 1 vs. 85 ± 1%, p < 0.05). Glibenclamide administration also reduced HT based on ECASS criteria, densitometry (0.94 ± 0.1 vs. 1.15 ± 0.2, p < 0.01), and quantitative hemoglobin concentration (2.7 ± 1.5 vs. 6.2 ± 4.6 uL, p = 0.011). In the second cohort with rt-PA coadministration, concordant effects on HT were observed with glibenclamide. CONCLUSIONS: Taken together, these studies demonstrated that glibenclamide reduced the amount of edema and HT after severe ischemia. This study suggests that co-administration of glibenclamide may be worth further study in severe stroke patients treated with EVT with or without IV rt-PA.

Time-dependent, dynamic prediction of fatty acid-binding protein 4, Galectin-3, and soluble ST2 measurement with poor outcome after acute stroke.

BACKGROUND: Time-dependent change in the level of biomarkers after stroke is not well understood. We sought to compare fatty acid-binding protein 4 (FABP4), Galectin-3, and soluble ST2 to ascertain for a change in prediction of outcome at admission and 48 h later. METHODS: Plasma FABP4, Galectin-3, and soluble ST2 were measured in biospecimens from acute stroke patients at the time of admission (n = 383) and 48 h later (n = 244). Functional outcome was assessed at 90 days using the modified Rankin Scale and dichotomized into good (modified Rankin Scale 0-2) and poor outcome (modified Rankin Scale 3-6). RESULTS: On admission, elevated levels of each biomarker predicted poor outcome (FABP4: OR 1.92, 95% CI 1.42-2.59, P < 0.0001; Galectin-3: OR 1.85, 95% CI 1.42-2.40, P < 0.0001; soluble ST2: OR 1.55, 95% CI 1.22-1.97, P < 0.0001) and death (FABP4: OR 2.45; 95% CI 1.51-3.98; P < 0.0001; Galectin-3: OR 2.12; 95% CI 1.50-3.30; P < 0.0001; soluble ST2: OR 2.17; 95% CI 1.58-2.99; P < 0.0001). At 48 h, soluble ST2 predicted poor outcome (OR 2.62, 95% CI 1.77-3.88, P < 0.0001) and mortality (OR 3.36, 95% CI 2.06-5.48, P < 0.0001), and Galectin-3 predicted mortality only (OR 1.81, 95% CI 1.05-3.10, P = 0.033). FABP4 measured at 48 h was not predictive of outcome or death. Associations of Galectin-3 and soluble ST2 with outcome or mortality were independent of age, sex, and NIHSS, whereas those with FABP4 were not. CONCLUSIONS: Galectin-3 performed better when measured on admission, whereas soluble ST2 was predictive at admission and better at 48 h after stroke. The time-dependent differences may reflect the evolving role of these pathways after acute stroke.

CD163 deficiency increases foam cell formation and plaque progression in atherosclerotic mice.

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/- /CD163-/- compared with ApoE-/- /CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression.

Soluble ST2 links inflammation to outcome after subarachnoid hemorrhage.

OBJECTIVE: To investigate whether soluble growth stimulation expressed gene 2 (sST2), a prognostic marker in cardiovascular and inflammatory disorders, is associated with neurological injury after aneurysmal subarachnoid hemorrhage (SAH). METHODS: We studied SAH patients from 2 independent cohorts. Outcome assessments included functional status at 90 days using the modified Rankin Scale (mRS), mortality, and delayed cerebral ischemia (DCI). The relationships between sST2 plasma level and outcome measures were assessed in both cross-sectional and longitudinal analysis. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry. RESULTS: In the discovery cohort, sST2 predicted 90-day mRS 3-6 (C index = 0.724, p < 0.001) and mortality in Kaplan-Meier analysis (p < 0.001). The association with functional status was independent of age, sex, World Federation of Neurosurgical Societies score, modified Fisher score, treatment modality, and cardiac comorbidities (adjusted odds ratio = 2.28, 95% confidence interval = 1.04-5.00, p = 0.039). Higher sST2 concentration was observed in those patients with DCI (90.8 vs 53.7ng/ml, p = 0.003). These associations were confirmed in a replication cohort. In patients with high sST2, flow cytometry identified decreased expression of CD14 (4.27 × 105 ± 2,950 arbitrary unit (AU) vs 5.64 × 105 ± 1,290 AU, p < 0.001), and increased expression of CD16 (39,960 ± 272 AU vs 34,869 ± 183 AU, p < 0.001). INTERPRETATION: Plasma sST2 predicts DCI, functional outcome, and mortality after SAH, independent of clinical and radiographic markers. Elevated sST2 is also associated with changes in CD14+ CD16+ monocytes. ANN NEUROL 2019;86:384-394.

Soluble ST2 predicts outcome and hemorrhagic transformation after acute stroke.

OBJECTIVE: ST2 is a member of the toll-like receptor superfamily that can alter inflammatory signaling of helper T-cells. We investigated whether soluble ST2 (sST2) could independently predict outcome and hemorrhagic transformation (HT) in the setting of stroke. METHODS: We measured sST2 in patients enrolled in the Specialized Program of Translational Research in Acute Stroke (SPOTRIAS) network biomarker study. 646 patients had plasma samples collected at the time of hospital admission and 210 patients had a second sample collected 48 h after stroke onset. Functional outcome was assessed using the modified Rankin Scale (mRS), with good and poor outcomes defined as mRS 0-2 and 3-6, respectively. HT was classified using ECASS criteria. The relationships between sST2, outcome, and HT were evaluated using multivariable logistic regression, Kaplan-Meier survival analysis and receiver operating characteristic curves. RESULTS: 646 patients were included in the analysis (mean age 69 years; 44% women), with a median NIHSS of 5 [IQR: 2-12]. The median sST2 level on hospital admission was 35.0 ng/mL [IQR: 25.7-49.8 ng/mL] and at 48 h it was 37.4 ng/mL [IQR 27.9-55.6 ng/mL]. sST2 was independently associated with poor outcome (OR: 2.77, 95% CI: 1.54-5.06; P = 0.003) and mortality (OR: 3.56, 95% CI: 1.58-8.38, P = 0.001) after multivariable adjustment. Plasma sST2 was also associated with hemorrhagic transformation after adjustment for traditional risk factors (OR: 5.58, 95% CI: 1.40-37.44, P = 0.039). INTERPRETATION: Soluble ST2 may serve as a prognostic biomarker for outcome and hemorrhagic transformation in patients with acute stroke. ST2 may link neuroinflammation and secondary injury after stroke.

TWEAK blockade decreases atherosclerotic lesion size and progression through suppression of STAT1 signaling in diabetic mice.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK/Tnfsf12) is a cytokine implicated in different steps associated with vascular remodeling. However, the role of TWEAK under hyperglycemic conditions is currently unknown. Using two different approaches, genetic deletion of Tnfsf12 and treatment with a TWEAK blocking mAb, we have analyzed the effect of TWEAK inhibition on atherosclerotic plaque progression and stability in streptozotocin-induced diabetic ApoE deficient mice. Genetic inactivation of Tnfsf12 reduced atherosclerosis extension and severity in diabetic ApoE deficient mice. Tnfsf12 deficient mice display a more stable plaque phenotype characterized by lower lipid and macrophage content within atherosclerotic plaques. A similar phenotype was observed in diabetic mice treated with anti-TWEAK mAb. The proatherosclerotic effects of TWEAK were mediated, at least in part, by STAT1 activation and expression of proinflammatory target genes (CCL5, CXCL10 and ICAM-1), both in plaques of ApoE mice and in cultured vascular smooth muscle cells (VSMCs) under hyperglycemic conditions. Loss-of-function experiments demonstrated that TWEAK induces proinflammatory genes mRNA expression through its receptor Fn14 and STAT1 activation in cultured VSMCs. Overall, TWEAK blockade delay plaque progression and alter plaque composition in diabetic atherosclerotic mice. Therapies aimed to inhibit TWEAK expression and/or function could protect from diabetic vascular complications.

Soluble TWEAK and Major Adverse Cardiovascular Events in Patients with CKD.

BACKGROUND AND OBJECTIVES: Soluble TNF-like weak inducer of apoptosis (sTWEAK) is a proinflammatory cytokine belonging to the TNF superfamily. sTWEAK concentrations have been associated with the presence of CKD and cardiovascular disease (CVD). We hypothesized that sTWEAK levels may relate to a higher prevalence of atherosclerotic plaques, vascular calcification, and cardiovascular outcomes observed in patients with CKD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A 4-year prospective, multicenter, longitudinal study was conducted in 1058 patients with CKD stages 3-5D (mean age =58±13 years old; 665 men) but without any history of CVD from the NEFRONA Study (a study design on the prevalence of surrogate markers of CVD). Ankle-brachial index and B-mode ultrasound were performed to detect the presence of carotid and/or femoral atherosclerotic plaques together with biochemical measurements and sTWEAK assessment. Patients were followed for cardiovascular outcomes (follow-up of 3.13±1.15 years). RESULTS: Patients with more advanced CKD had lower sTWEAK levels. sTWEAK concentrations were independently and negatively associated with carotid intima-media thickness. sTWEAK levels were lower in patients with carotid atherosclerotic plaques but not in those with femoral plaques. After adjustment by confounders, the odds ratio (OR) for presenting carotid atherosclerotic plaques in patients in the lowest versus highest tertile of sTWEAK was 4.18 (95% confidence interval [95% CI], 2.89 to 6.08; P<0.001). Furthermore, sTWEAK levels were lower in patients with calcified carotid atherosclerotic plaques. The OR for presenting calcified carotid plaques was 1.77 (95% CI, 1.06 to 2.93; P=0.02) after multivariable adjustment. After the follow-up, 41 fatal and 68 nonfatal cardiovascular events occurred. In a Cox model, after controlling for potential confounding factors, patients in the lowest tertile of sTWEAK concentrations had a higher risk of fatal and nonfatal cardiovascular events (hazard ratio [HR], 2.40; 95% CI, 1.33 to 4.33; P=0.004) and cardiovascular mortality (HR, 2.67; 95% CI, 1.05 to 6.76; P=0.04). CONCLUSIONS: Low sTWEAK levels were associated with the presence of carotid atherosclerotic plaques in patients with CKD. Additionally, lower sTWEAK levels were associated with a higher risk of cardiovascular morbidity and mortality.

TWEAK/Fn14 interaction promotes oxidative stress through NADPH oxidase activation in macrophages.

AIM: The interaction between TNF-like weak inducer of apoptosis (TWEAK, Tnfsf12) and the receptor, fibroblast growth factor-inducible 14 (Fn14), regulates vascular damage through different mechanisms, including inflammation. Oxidative stress plays a major role in inflammation and the development of atherosclerosis, but the relationship between TWEAK and oxidative stress is, however, poorly understood. METHODS AND RESULTS: In this study, we found that TWEAK and Fn14 are co-localized with the NADPH subunits, p22phox and Nox2, in human advanced atherosclerotic plaques. Using primary human macrophages and a murine macrophage cell line, we demonstrate that TWEAK promotes ROS production and enhances NADPH oxidase activity. Hence, we show a direct involvement of the TWEAK-Fn14 axis in oxidative stress, as genetic silencing of Fn14 or Nox2 abrogates the TWEAK-induced ROS production. Furthermore, our results point at Rac1 as an upstream mediator of TWEAK during oxidative stress. Finally, using an in vivo murine model we confirmed the major role of TWEAK in oxidative stress, as genetic silencing of Tnfsf12 in an ApoE(-/-) background reduces the number of DHE and 8-hydroxydeoxyguanosine-positive macrophages by 50%. CONCLUSIONS: Our results suggest that TWEAK regulates vascular damage by stimulating ROS production in an Nox2-dependent manner. These new insights into the TWEAK/Fn14 axis underline their potential use as therapeutic targets in atherosclerosis.

Soluble TWEAK levels predict the presence of carotid atherosclerotic plaques in subjects free from clinical cardiovascular diseases.

OBJECTIVE: Reduced soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) levels have been related with cardiovascular disease. However, there are no data on the relationship between sTWEAK and atherosclerotic burden in subjects with or without cardiovascular risk factors but free from clinical disease. We have analyzed the association between circulating sTWEAK levels and the presence of carotid and/or femoral atherosclerotic plaques in subjects without known vascular disease. METHODS: A multicenter, cross-sectional study was conducted in 448 subjects free from clinical CVD. B-mode ultrasound was performed to detect the presence of carotid and/or femoral atherosclerotic plaques. sTWEAK concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: sTWEAK serum levels were reduced in parallel with an increment in cardiovascular risk factors. sTWEAK concentrations were independently and negatively associated with carotid intima/media thickness. Subjects with atherosclerotic plaques showed a reduction in sTWEAK levels [808 (645-963) vs 993 (830-1278); p < 0.001]. A gradual decrease in sTWEAK levels was observed as the number of atherosclerotic plaques increased in our studied population. When we analyzed sTWEAK levels according to the vascular territory affected, we observed that sTWEAK concentrations were only diminished in subjects with carotid atherosclerotic plaques but not in those with femoral plaques. Following adjustment for various confounders, the OR for presenting carotid atherosclerotic plaque in subjects in lower vs higher tertile of sTWEAK levels was 8.09 [4.30-15.23; median (IQR); p < 0.001]. CONCLUSIONS: Diminished sTWEAK concentrations were significantly and independently associated with the presence of carotid atherosclerotic plaques in asymptomatic subjects.

La atorvastatina inhibe la progresión de la lesión aterosclerótica inducida por el factor inductor débil de apoptosis similar al factor de necrosis tumoral en ratones deficientes en apolipoproteína E / Atorvastatin inhibits the atherosclerotic lesion induced by tumor necrosis factor-like weak inducer of apoptosis in apolipoprotein E deficient mice

Objetivos: La interacción del factor inductor débil de apoptosis similar al factor de necrosis tumoral (TWEAK) con su receptor Fn14 acelera el desarrollo de la lesión aterosclerótica en ratones deficientes en ApoE (ApoE KO). En este trabajo hemos analizado el efecto de un inhibidor de la HMG-CoA reductasa, la atorvastatina, sobre el desarrollo de la lesión aterosclerótica acelerada por TWEAK en ratones ApoE KO. Materiales y métodos: Se alimentaron ratones ApoE KO de 8 semanas de edad durante 4 semanas con una dieta hiperlipidémica y se aleatorizaron en 3 grupos: ratones tratados i.p. con salino (control), tratados con TWEAK recombinante (10 μg/kg/2 veces a la semana) o tratados con TWEAK recombinante más atorvastatina (1 mg/kg/día) durante 4 semanas más. Se analizó el tamaño, la composición celular, la respuesta inflamatoria y la expresión de Fn14 en las lesiones ateroscleróticas presentes en la raíz aórtica de los ratones. Resultados: La inyección sistémica de TWEAK aumentó el tamaño de la lesión y el cociente colágeno/lípidos así como la respuesta inflamatoria asociada a un aumento en la actividad de NF-κB, expresión de MCP-1 y RANTES y a una mayor presencia de macrófagos en las placas ateroscleróticas de ratones ApoE KO. El tratamiento con atorvastatina fue capaz de prevenir los cambios inducidos por TWEAK en las lesiones ateroscleróticas. Finalmente, el tratamiento con atorvastatina disminuyó la expresión de Fn14 en las lesiones ateroscleróticas de ratones ApoE KO. Conclusiones: La atorvastatina previene los efectos proaterogénicos inducidos por TWEAK en el ratón ApoE KO, efecto relacionado con la inhibición de la expresión de Fn14. Estos resultados aportan nueva información sobre los efectos beneficiosos de las estatinas en el tratamiento de las enfermedades cardiovasculares

Aim: Interaction of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) with its receptor Fn14 accelerates atherosclerotic plaque development in ApoE deficient mice (ApoE KO). In this work, an analysis has been made on the effect of an HMG-CoA reductase inhibitor, atorvastatin, on atherosclerotic plaque development accelerated by TWEAK in ApoE KO mice. Materials and methods: Eight week-old ApoE KO mice were fed with a high cholesterol diet for 4 weeks. The animals were then randomized into 3 groups: mice injected i.p. with saline, recombinant TWEAK (10 μg/kg/twice a week), or recombinant TWEAK plus atorvastat in (1 mg/kg/day) for 4 weeks. The lesion size, cellular composition, lipid and collagen content were analyzed, as well as inflammatory response in atherosclerotic plaques present in aortic root of mice. Results: TWEAK treated mice showed an increase in atherosclerotic plaque size, as well as in collagen/lipid ratio compared with control mice. In addition, macrophage content, MCP-1 and RANTES expression, and NF-κB activation were augmented in atherosclerotic plaques present in aortic root of TWEAK treated mice compared with control mice. Treatment with atorvastatin prevented all these changes induced by TWEAK in atherosclerotic lesions. Atorvastatin treatment also decreased Fn14 expression in the atherosclerotic plaques of ApoE KO mice. Conclusions: Atorvastatin prevents the pro-atherogenic effects induced by TWEAK in ApoE KO mice, which could be related to the inhibition of Fn14 expression. The results of this study provide new information on the beneficial effects of statin treatment in cardiovascular diseases

[Atorvastatin inhibits the atherosclerotic lesion induced by tumor necrosis factor-like weak inducer of apoptosis in apolipoprotein E deficient mice]. / La atorvastatina inhibe la progresión de la lesión aterosclerótica inducida por el factor inductor débil de apoptosis similar al factor de necrosis tumoral en ratones deficientes en apolipoproteína E.

AIM: Interaction of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) with its receptor Fn14 accelerates atherosclerotic plaque development in ApoE deficient mice (ApoE KO). In this work, an analysis has been made on the effect of an HMG-CoA reductase inhibitor, atorvastatin, on atherosclerotic plaque development accelerated by TWEAK in ApoE KO mice. MATERIALS AND METHODS: Eight week-old ApoE KO mice were fed with a high cholesterol diet for 4 weeks. The animals were then randomized into 3 groups: mice injected i.p. with saline, recombinant TWEAK (10 µg/kg/twice a week), or recombinant TWEAK plus atorvastatin (1 mg/kg/day) for 4 weeks. The lesion size, cellular composition, lipid and collagen content were analyzed, as well as inflammatory response in atherosclerotic plaques present in aortic root of mice. RESULTS: TWEAK treated mice showed an increase in atherosclerotic plaque size, as well as in collagen/lipid ratio compared with control mice. In addition, macrophage content, MCP-1 and RANTES expression, and NF-κB activation were augmented in atherosclerotic plaques present in aortic root of TWEAK treated mice compared with control mice. Treatment with atorvastatin prevented all these changes induced by TWEAK in atherosclerotic lesions. Atorvastatin treatment also decreased Fn14 expression in the atherosclerotic plaques of ApoE KO mice. CONCLUSIONS: Atorvastatin prevents the pro-atherogenic effects induced by TWEAK in ApoE KO mice, which could be related to the inhibition of Fn14 expression. The results of this study provide new information on the beneficial effects of statin treatment in cardiovascular diseases.

Tumor necrosis factor-like weak inducer of apoptosis or Fn14 deficiency reduce elastase perfusion-induced aortic abdominal aneurysm in mice.

BACKGROUND: Abdominal aortic aneurysm (AAA) involves leukocyte recruitment, inflammatory cytokine production, vascular cell apoptosis, neovascularization, and vascular remodeling, all of which contribute to aortic dilatation. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a cytokine implicated in proinflammatory responses, angiogenesis, and matrix degradation but its role in AAA formation is currently unknown. METHODS AND RESULTS: Experimental AAA with aortic elastase perfusion in mice was induced in wild-type (WT), TWEAK deficient (TWEAK KO), or Fn14-deficient (Fn14 KO) mice. TWEAK or Fn14 KO deficiency reduced aortic expansion, lesion macrophages, CD3(+) T cells, neutrophils, CD31(+) microvessels, CCL2 and CCL5 chemokines expression, and MMP activity after 14 days postperfusion. TWEAK and Fn14 KO mice also showed a reduced loss of medial vascular smooth muscle cells (VSMC) that was related to a reduced number of apoptotic cells in these animals compared with WT mice. Aortas from WT animals present a higher disruption of the elastic layer and MMP activity than those from TWEAK or Fn14 KO mice, indicating a diminished vascular remodeling in KO animals. In vitro experiments unveiled that TWEAK induces CCL5 secretion and MMP-9 activation in both VSMC and bone marrow-derived macrophages, and decrease VSMC viability, effects dependent on Fn14. CONCLUSIONS: TWEAK/Fn14 axis participates in AAA formation by promoting lesion inflammatory cell accumulation, angiogenesis, matrix-degrading protease expression, and vascular remodeling. Blocking TWEAK/Fn14 interaction could be a new target for the treatment of AAA.

Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice.

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12(-/-) ApoE(-/-) ) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12(+/+) ApoE(-/-) control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) . A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12(+/+) ApoE(-/-) mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treatment in TNFSF12(+/+) ApoE(-/-) mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.

HMGB1 expression and secretion are increased via TWEAK-Fn14 interaction in atherosclerotic plaques and cultured monocytes.

OBJECTIVE: High-mobility group box 1 (HMGB1), a DNA-binding cytokine expressed mainly by macrophages, contributes to lesion progression and chronic inflammation within atherosclerotic plaque. It has been suggested that different cytokines could regulate HMGB1 expression in monocytes. We have analyzed the effect of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) on HMGB1 expression both in vivo and in vitro. METHODS AND RESULTS: Expression of TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) was positively correlated with HMGB1 in human carotid atherosclerotic plaques. TWEAK increased HMGB1 mRNA expression and protein secretion in human acute monocytic leukemia cell line cultured monocytes. TWEAK-mediated HMGB1 increase was only observed in M1 macrophages but not in M2 ones. These effects were reversed using blocking anti-Fn14 antibody or nuclear factor-kappa B and phosphotidylinositol-3 kinase inhibitors. TWEAK also increased monocyte chemoattractant protein-1 secretion in human acute monocytic leukemia cell line cells, an effect blocked with an HMGB1 small interfering RNA. Systemic TWEAK injection in ApoE(-/-) mice increased HMGB1 protein expression in the aortic root and mRNA expression in total aorta of ApoE(-/-) mice. Conversely, TWEAK-blocking antibodies diminished HMGB1 protein and mRNA expression compared with IgG-treated mice. CONCLUSIONS: Our results indicate that TWEAK can regulate expression and secretion of HMGB1 in monocytes/macrophages, participating in the inflammatory response associated with atherosclerotic plaque development.

Hyperlipidemia-associated renal damage decreases Klotho expression in kidneys from ApoE knockout mice.

BACKGROUND: Klotho is a renal protein with anti-aging properties that is downregulated in conditions related to kidney injury. Hyperlipidemia accelerates the progression of renal damage, but the mechanisms of the deleterious effects of hyperlipidemia remain unclear. METHODS: We evaluated whether hyperlipidemia modulates Klotho expression in kidneys from C57BL/6 and hyperlipidemic apolipoprotein E knockout (ApoE KO) mice fed with a normal chow diet (ND) or a Western-type high cholesterol-fat diet (HC) for 5 to 10 weeks, respectively. RESULTS: In ApoE KO mice, the HC diet increased serum and renal cholesterol levels, kidney injury severity, kidney macrophage infiltration and inflammatory chemokine expression. A significant reduction in Klotho mRNA and protein expression was observed in kidneys from hypercholesteromic ApoE KO mice fed a HC diet as compared with controls, both at 5 and 10 weeks. In order to study the mechanism involved in Klotho down-regulation, murine tubular epithelial cells were treated with ox-LDL. Oxidized-LDL were effectively uptaken by tubular cells and decreased both Klotho mRNA and protein expression in a time- and dose-dependent manner in these cells. Finally, NF-κB and ERK inhibitors prevented ox-LDL-induced Klotho downregulation. CONCLUSION: Our results suggest that hyperlipidemia-associated kidney injury decreases renal expression of Klotho. Therefore, Klotho could be a key element explaining the relationship between hyperlipidemia and aging with renal disease.

Targeting chemokines in proteinuria-induced renal disease.

INTRODUCTION: Proteinuria is a common finding in glomerular diseases that contributes to the progression of chronic kidney injury. Tubular cells reabsorb the excess of albumin and other plasma proteins from the tubular lumen, triggering several pathophysiologic responses, such as overexpression of fibrogenic mediators and inflammatory chemokines. Chemokines are implicated both in the recruitment of inflammatory infiltrate and in a number of physiological and pathological processes related to protein overload. AREAS COVERED: In recent years, the specific chemokines and their receptors and the intracellular signaling pathways involved in proteinuria-induced renal damage have been identified. This review provides an overview of the role of chemokines and their receptors in proteinuria-related renal disease and summarizes novel therapeutic approaches to restrain the progression of renal damage. EXPERT OPINION: Inhibition of chemokine-induced biological activities is a promising therapeutic strategy in proteinuric disorders. Neutralizing antibodies and small organic molecules targeting chemokines and chemokine receptors have been proven to prevent inflammation and renal damage in experimental models of protein overload. Some of these compounds are currently being tested in human clinical trials.

TWEAK-Fn14 interaction enhances plasminogen activator inhibitor 1 and tissue factor expression in atherosclerotic plaques and in cultured vascular smooth muscle cells.

AIMS: atherosclerotic plaque development can conclude with a thrombotic acute event triggered by plaque rupture/erosion. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumour necrosis factor superfamily that, through its receptor, fibroblast growth factor-inducible 14 (Fn14), participates in vascular remodelling, increasing vascular inflammatory responses and atherosclerotic lesion size in ApoE knockout mice. However, the role of the TWEAK-Fn14 axis in thrombosis has not been previously investigated. METHODS AND RESULTS: we have examined whether TWEAK regulates expression of prothrombotic factors such as tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) in atherosclerotic plaques as well as in human aortic vascular smooth muscle cells (hASMCs) in culture. Expression of TF and PAI-1 was colocalized and positively correlated with Fn14 in human carotid atherosclerotic plaques. In vitro, TWEAK increased TF and PAI-1 mRNA, protein expression and activity in hASMCs. All these effects were reversed using blocking anti-TWEAK monoclonal antibody, anti-Fn14 antibody or Fn14 small interfering RNA, indicating that TWEAK increased the prothrombotic state through its receptor, Fn14. Finally, ApoE(-/-) mice were fed a hyperlipidaemic diet for 10 weeks, then randomized and treated with saline (controls), TWEAK (10 microg/kg/day), anti-TWEAK neutralizing monoclonal antibody (1000 µg/kg/day), or non-specific immunoglobulin G (1000 microg/kg/day) daily for 9 days. Systemic TWEAK injection increased TF and PAI-1 protein expression in the aortic root of ApoE(-/-) mice. Conversely, TWEAK blocking antibodies diminished both TF and PAI-1 protein expression compared with non-specific immunoglobulin G-treated mice. CONCLUSIONS: our results indicate that the TWEAK-Fn14 axis can regulate activation of TF and PAI-1 expression in vascular cells. TWEAK-Fn14 may be a therapeutic target in the prothrombotic complications associated with atherosclerosis.