Globular adiponectin counteracts VCAM-1-mediated monocyte adhesion via AdipoR1/NF-κB/COX-2 signaling in human aortic endothelial cells

Abstract

Adiponectin (Ad) is an insulin-sensitizing adipocytokine with anti-inflammatory and vasoprotective properties. Cleavage of native full-length Ad (fAd) by elastases from activated monocytes generates globular Ad (gAd). Increased gAd levels are observed in the proximity of atherosclerotic lesions, but the physiological meaning of this proteolytic Ad fragment in the cardiovascular system is controversial. We compared molecular and biological properties of fAd and gAd in human aortic endothelial cells (HAEC). In control HAEC, both fAd and gAd acutely stimulated nitric oxide (NO) production by AMPK-dependent pathways. With respect to fAd, gAd more efficiently increased activation of NF-κB signaling pathways, resulting in cyclooxygenase-2 (COX-2) overexpression and COX-2-dependent prostacyclin 2 (PGI(2)) release. In contrast with fAd, gAd also increased p38 MAPK phosphorylation and VCAM-1 expression, ultimately enhancing adhesion of monocytes to endothelial cells. In HAEC lacking AdipoR1 (by siRNA), both activation of NF-κB as well as COX-2 overexpression by gAd were abrogated. Conversely, gAd-mediated p38MAPK activation and VCAM-1 expression were unaffected, and monocyte adhesion was greatly enhanced. In HAEC lacking COX-2 (by siRNA), reduced levels of PGI(2) further increased gAd-dependent monocyte adhesion. Our findings suggest that biological activities of fAd and gAd in endothelium do not completely overlap, with gAd possessing both AdipoR1-dependent ability to stimulate COX-2 expression and AdipoR1-independent effects related to expression of VCAM-1 and adhesion of monocytes to endothelium.

Fig. 1.

Overlapping and distinct effects of globular (gAd) and full-length adiponectin (fAd) in human aortic endothelial cells (HAEC). A: electrophoresis mobility and oligomerization status of gAd and fAd. Fifty microliters of conditioned media (CM) collected from HAEC treated with fAd or gAd (10 μg/ml, 1 h) was loaded on a 12% nonreducing SDS-PAGE, and membranes were probed with anti-adiponectin antibody. A 90-kDa molecular mass band was found in the CM of fAd-treated HAEC cells. Conversely, a 19-kDa molecular mass band was detected in HAEC treated with gAd. B: HAEC were serum starved (3 h) and loaded with the nitric oxide (NO)-specific fluorescent dye 4,5-diaminofluorescein diacetate (3 μM; Cayman Chemical, Ann Arbor, MI) in the absence or presence of NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME; 100 μM). HAEC were then stimulated with fAd or gAd (10 mg/ml, 10 min), fixed in 2% paraformaldehyde (PFA) for 5 min at 4°C, and then viewed using an epifluorescent microscope. Emission of green light (510 nm) from cells excited at 480 nm is indicative of NO production. Representative images are shown for experiments that were repeated independently 3 times. C: lysates from HAEC stimulated with fAd or gAd (10 mg/ml, 10 min) were subjected to immunoblotting for phosphorylated (ph) and total forms of Akt and AMP-activated protein kinase (AMPK) proteins. Both fAd and gAd increased phosphorylation levels of Akt and AMPK. D: time course experiments (0, 15, 30, 45, and 60 min) indicate that increased phosphorylation of p65, IκBα, and p38 MAPK in response to treatment with gAd peaks after 1-h stimulation. Treatment with fAd did not significantly enhance phosphorylation levels for NF-κB or p38 MAPK at any of the time points considered.

Fig. 2.

Differential activation of proinflammatory signaling by gAd and fAd in HAEC. HAEC cultured as described in materials and metthods were treated with TNFα (10 ng/ml), fAd (10 μg/ml), or gAd (10 μg/ml) for the indicated time points. HAEC lysates were subjected to immunoblotting with antibodies for total and phosphorylated forms of p42/44 MAPK, p38 MAPK, p65, and IκBα. A: representative immunoblots for results obtained in HAEC stimulated with TNFα (10 ng/ml), fAd (10 μg/ml), or gAd (10 μg/ml) for 10 min. Each bar represents the mean ± SE of densitometric analysis for phosphorylated proteins normalized to their respective total forms. B: representative immunoblots for results obtained in HAEC stimulated with TNFα (10 ng/ml) for 10 min and fAd (10 μg/ml) or gAd (10 μg/ml) for 1 h. Each bar represents the mean ± SE of densitometric analysis for phosphorylated proteins normalized to their respective total forms. C: experiments described in B were repeated in the absence or presence of NF-κB inhibitor BAY 11-7082 (20 μM, 1-h preincubation) or p38 MAPK inhibitor SB-203580 (10 μM, 1-h preincubation). Representative immunoblots from at least 3 independent experiments are shown for each condition. Each bar represents the mean ± SE of densitometric analysis for phosphorylated proteins normalized to their respective total forms. *P < 0.05, **P < 0.01 vs. basal (Bas) conditions; †P < 0.05, ††P < 0.01 vs. TNFα; ‡P < 0.05, ‡‡P < 0.01 vs. gAd.

Fig. 3.

gAd induces overexpression of cyclooxygenase-2 (COX-2) by p38 MAPK and NF-κB pathways. A: mRNA levels of COX-2 detected in HAEC treated for 8 h with TNFα (10 ng/ml), fAd, or gAd (10 μg/ml). B: protein concentrations of 6-keto-prostaglandin F1α (6-keto-PGF-1α) in CM from HAEC treated as in A. C: levels of COX-2 protein expression in lysates of HAEC treated as in A in the absence or presence of NF-κB inhibitor BAY 11-7082 (20 μM, 1-h preincubation), p38 MAPK inhibitor SB-203580 (10 μM, 1-h preincubation), or p42/44 MAPK inhibitor PD-98059 (20 μM, 1-h preincubation). Representative immunoblots from at least 3 independent experiments are shown. Bar graphs indicate the mean ± SE of densitometric analysis for COX-2 protein normalized to β-actin expression. *P < 0.05, **P < 0.01 vs. basal; ††P < 0.01 vs. TNFα; ‡P < 0.05 vs. gAd.

Fig. 4.

gAd induces overexpression of vascular cell adhesion molecule-1 (VCAM-1) by p38 MAPK and NF-κB pathways. A: mRNA levels of VCAM-1 detected in HAEC treated for 8 h with TNFα (10 ng/ml), fAd, or gAd (10 μg/ml). B: soluble VCAM-1 protein in CM from HAEC treated as in A. C: levels of VCAM-1 protein expression in lysates of HAEC treated as in A in the absence or presence of NF-κB inhibitor BAY 11-7082 (20 μM, 1-h preincubation), p38 MAPK inhibitor SB-203580 (10 μM, 1-h preincubation), or p42/44 MAPK inhibitor PD-98059 (20 μM, 1-h preincubation). Representative immunoblots from at least 3 independent experiments are shown. Bar graphs indicate the mean ± SE of densitometric analysis for VCAM-1 protein normalized to β-actin expression. **P < 0.01 vs. basal; ‡P < 0.05; ‡‡P < 0.01 vs. gAd.

Fig. 5.

gAd- and TNFα-enhanced expression of COX-2 and VCAM-1 in HAEC. A: HAEC were left untreated or stimulated with TNFα (100 fg/ml to 100 ng/ml) or gAd (10 μg/ml) for 8 h. Lysates were subjected to immunoblotting for COX-2 or VCAM-1, and conditioned medium was analyzed for prostacyclin content. Representative immunoblots are shown for experiments that were independently repeated 3 times. B: confocal fluorescence images in the xy and xz planes showing immunolocalization of VCAM-1 protein (red fluorescence) and U937 human monocytes (green fluorescence) in mock transfected HAEC subjected to adhesion assay. Arrows in the xz scans indicate VCAM-1 protein surrounding monocytes at the level of plasma membrane. A similar pattern is shown for membrane-located VCAM-1 in HAEC stimulated with either 10 ng/ml TNFα or 10 μg/ml gAd. C: gAd- and fAd-dependent COX-2 expression. Representative blot shows protein levels of COX-2 in HAEC treated for 8 h with 10 and 30 μg/ml gAd and fAd. Bar graphs indicate the mean ± SE of densitometric analysis for VCAM-1 and COX-2 protein normalized to β-actin expression. *P < 0.05, **P < 0.01 vs. basal; †P < 0.05 vs. fAd.

Fig. 6.

Increased VCAM-1 expression correlates with enhanced monocyte adhesion to endothelial cells. A: HAEC cultured as described in materials and methods were left untreated or stimulated for 8 h with TNFα (10 ng/ml) or gAd (10 μg/ml) in the absence (top) or presence of p38 inhibitor SB-203580, COX-2 inhibitor NS-398, VCAM-1-neutralizing antibody, or Ad-neutralizing antibody. HAEC were then incubated for 1 h with calcein green-labeled U937 human monocytes and fixed in 1% PFA. Overlapping of bright-field images with green fluorescent images shows U937 monocytes over HAEC for each tested condition. Bar graphs indicate the mean ± SE of U937/HAEC ratio for experiments independently repeated at least 3 times. ADPN, adiponectin.

Fig. 7.

Abrogation of adiponectin receptor 1 (AdipoR1) or COX-2 enhances VCAM-1-mediated monocyte adhesion in response to gAd. A: HAEC cultured as described in materials and methods were transfected with scrambled siRNA, AdipoR1, or COX-2 siRNA. Forty-eight hours after transfection, HAEC were left untreated or stimulated for 8 h with TNFα (10 ng/ml) or gAd (10 μg/ml), incubated for 1 h with calcein green-labeled U937 human monocytes, and then fixed in 1% PFA. Overlapping of bright-field images with green fluorescent images shows U937 monocytes over HAEC for each tested condition. Bar graphs indicate the mean ± SE of U937/HAEC ratio for experiments independently repeated at least 3 times. B: mRNA levels of AdipoR1 and COX-2 detected in mock-transfected or specific siRNA-transfected HAEC under basal conditions and after stimulation with TNFα (10 ng/ml) or gAd (10 μg/ml). C: HAEC treated as described in A were stimulated with fAd (10 μg/ml) and gAd (10 μg/ml) for 8 h and lysates subjected to immunoblotting for endothelial nitric oxide synthase (eNOS; control), VCAM-1, or COX-2 expression. Representative immunoblots are shown for experiments that were independently repeated 3 times. D: protein concentrations of 6-keto-PGF-1α in the CM from mock-transfected and siCOX-2-transfected HAEC under basal conditions and after stimulation for 8 h with TNFα or gAd. Bar graphs indicate the mean ± SE of at least 3 independent experiments. *P < 0.05, **P < 0.01 vs. basal; †P < 0.05, ††P < 0.01 vs. mock transfected cells.