Kyle Preston

Adiponectin Inhibits Tumor Necrosis Factor-α–Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Interleukin-19 decreases leukocyte-endothelial cell interactions by reduction in endothelial cell adhesion molecule mRNA stability

Vascular endothelial cell (EC) inflammation is a key event in the pathogenesis of multiple vascular diseases. We tested the hypothesis that interleukin-19 (IL-19), an anti-inflammatory Th2 interleukin, could have a direct anti-inflammatory effect on ECs to decrease inflammation. IL-19 can significantly decrease tumor necrosis factor (TNF)-α-driven intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mRNA and protein abundance in cultured human coronary artery ECs (P < 0.01). IL-19 treatment of ECs, but not monocytes, significantly reduced monocyte adhesion to EC monolayers (P < 0.01). In vivo, systemic administration of IL-19 could significantly reduce TNF-α-induced leukocyte rolling and adhesion in wild-type mice as assayed by intravital microscopy (P < 0.05). IL-19 does not reduce TNF-α-stimulated NF-κB activation in ECs but does decrease serine phosphorylation and cytoplasmic translocation of the mRNA stability factor HuR and significantly reduces stability of ICAM-1 and VCAM-1 mRNA (P < 0.01). These data are the first to report that IL-19 can reduce leukocyte-endothelial cell adhesion and the first to propose reduction in HuR-mediated mRNA stability of ICAM-1 and VCAM-1 as a mechanism. Expression of IL-19 by ECs may represent a protective mechanism to promote resolution of the vascular response to inflammation. Function of IL-19 outside of the immune system is a novel concept, suggesting that resident vascular cells can adopt a Th2 phenotype, and has important ramifications for numerous inflammatory diseases.

Caveolin-1 Deletion Prevents Hypertensive Vascular Remodeling Induced by Angiotensin II.

It has been proposed that membrane microdomains, caveolae, in vascular cells are critical for signal transduction and downstream functions induced by angiotensin II (AngII). We have tested our hypothesis that caveolin-1 (Cav1), a major structural protein of vascular caveolae, plays a critical role in the development of vascular remodeling by AngII via regulation of epidermal growth factor receptor and vascular endothelial adhesion molecule-1. Cav1−/− and control Cav+/+ mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension. On AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of aorta and coronary and renal arteries in Cav1+/+ mice compared with sham-operated Cav1+/+ mice. AngII-infused Cav1+/+ mice also showed a phenotype of cardiac hypertrophy with increased heart weight to body weight ratio compared with control Cav1+/+ mice. In contrast, Cav1−/− mice infused with AngII showed attenuation of vascular remodeling but not cardiac hypertrophy. Similar levels of AngII-induced hypertension were found in both Cav1+/+ and Cav1−/− mice as assessed by telemetry. In Cav1+/+ mice, AngII enhanced tyrosine-phosphorylated epidermal growth factor receptor staining in the aorta, which was attenuated in Cav1−/− mice infused with AngII. Enhanced Cav1 and vascular endothelial adhesion molecule-1 expression was also observed in aorta from AngII-infused Cav1+/+ mice but not in Cav1−/− aorta. Experiments with vascular cells further provided a potential mechanism for our in vivo findings. These data suggest that Cav1, and presumably caveolae, in vascular smooth muscle and the endothelium plays a critical role in vascular remodeling and inflammation independent of blood pressure or cardiac hypertrophy regulation.

Vascular ADAM17 (a Disintegrin and Metalloproteinase Domain 17) Is Required for Angiotensin II/β-Aminopropionitrile–Induced Abdominal Aortic AneurysmNovelty and Significance

Angiotensin II (AngII)-activated epidermal growth factor receptor (EGFR) has been implicated in abdominal aortic aneurysm (AAA) development. In vascular smooth muscle cells (VSMC), AngII activates EGFR via a metalloproteinase, a disintegrin and metallopeptidase domain 17 (ADAM17). We hypothesized that AngII-dependent AAA development would be prevented in mice lacking ADAM17 in VSMCs. To test this concept, control and VSMC ADAM17 deficient mice were co-treated with AngII and a lysyl oxidase inhibitor, β-aminopropionitrile, to induce AAA. We found that 52.4% of control mice did not survive due to aortic rupture. All other surviving control mice developed AAA and demonstrated enhanced expression of ADAM17 in the AAA lesions. In contrast, all AngII and β-aminopropionitrile-treated VSMC ADAM17 deficient mice survived and showed reduction in external/internal diameters (51%/28%, respectively). VSMC ADAM17 deficiency was associated with lack of EGFR activation, interleukin-6 induction, ER/oxidative stress and matrix deposition in the abdominal aorta of treated mice. However, both VSMC ADAM17 deficient and control mice treated with AngII and β-aminopropionitrile developed comparable levels of hypertension. Treatment of C57Bl/6 mice with an ADAM17 inhibitory antibody but not with control IgG also prevented AAA development. In conclusion, VSMC ADAM17 silencing or systemic ADAM17 inhibition appears to protect mice from AAA formation. The mechanism appears to involve suppression of EGFR activation.Angiotensin II (AngII)–activated epidermal growth factor receptor has been implicated in abdominal aortic aneurysm (AAA) development. In vascular smooth muscle cells (VSMCs), AngII activates epidermal growth factor receptor via a metalloproteinase, ADAM17 (a disintegrin and metalloproteinase domain 17). We hypothesized that AngII-dependent AAA development would be prevented in mice lacking ADAM17 in VSMCs. To test this concept, control and VSMC ADAM17-deficient mice were cotreated with AngII and a lysyl oxidase inhibitor, &bgr;-aminopropionitrile, to induce AAA. We found that 52.4% of control mice did not survive because of aortic rupture. All other surviving control mice developed AAA and demonstrated enhanced expression of ADAM17 in the AAA lesions. In contrast, all AngII and &bgr;-aminopropionitrile-treated VSMC ADAM17-deficient mice survived and showed reduction in external/internal diameters (51%/28%, respectively). VSMC ADAM17 deficiency was associated with lack of epidermal growth factor receptor activation, interleukin-6 induction, endoplasmic reticulum/oxidative stress, and matrix deposition in the abdominal aorta of treated mice. However, both VSMC ADAM17-deficient and control mice treated with AngII and &bgr;-aminopropionitrile developed comparable levels of hypertension. Treatment of C57Bl/6 mice with an ADAM17 inhibitory antibody but not with control IgG also prevented AAA development. In conclusion, VSMC ADAM17 silencing or systemic ADAM17 inhibition seems to protect mice from AAA formation. The mechanism seems to involve suppression of epidermal growth factor receptor activation.

Adiponectin Inhibits Tumor Necrosis Factor- -Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Mechanistic Role of the Calcium-Dependent Protease Calpain in the Endothelial Dysfunction Induced by MPO (Myeloperoxidase)Novelty and Significance

MPO (myeloperoxidase) is a peroxidase enzyme secreted by activated leukocytes that plays a pathogenic role in cardiovascular disease, mainly by initiating endothelial dysfunction. The molecular mechanisms of the endothelial damaging action of MPO remain though largely elusive. Calpain is a calcium-dependent protease expressed in the vascular wall. Activation of calpains has been implicated in inflammatory disorders of the vasculature. Using endothelial cells and genetically modified mice, this study identifies the µ-calpain isoform as novel downstream signaling target of MPO in endothelial dysfunction. Mouse lung microvascular endothelial cells were stimulated with 10 nmol/L MPO for 180 minutes. MPO denitrosylated µ-calpain C-terminus domain, and time dependently activated µ-calpain, but not the m-calpain isoform. MPO also reduced Thr172 AMPK (AMP-activated protein kinase) and Ser1177 eNOS (endothelial nitric oxide synthase) phosphorylation via upregulation of PP2A (protein phosphatase 2) expression. At the functional level, MPO increased endothelial VCAM-1 (vascular cell adhesion molecule 1) abundance and the adhesion of leukocytes to the mouse aorta. In MPO-treated endothelial cells, pharmacological inhibition of calpain activity attenuated expression of VCAM-1 and PP2A, and restored Thr172 AMPK and Ser1177 eNOS phosphorylation. Compared with wild-type mice, µ-calpain deficient mice experienced reduced leukocyte adhesion to the aortic endothelium in response to MPO. Our data first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO. The MPO/calpain/PP2A signaling pathway may provide novel pharmacological targets for the treatment of inflammatory vascular disorders.

Adiponectin Inhibits TNF-α-Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Adiponectin Inhibits Tumor Necrosis Factor-α–Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and ActivationNovelty and Significance

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.