Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo

Abstract

This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad(-/-)) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad(-/-) mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad(-/-) mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-alpha-induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with N(omega)-nitro-L-arginine methyl ester ( L-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.

Figure 1. Leukocyte-endothelium interactions in mouse peri-intestinal venules are increased by endogenous adiponectin deficiency.

(A) Leukocyte rolling flux in WT mice and Ad^–/– mice before and after treatment with human recombinant gAd (25 μg, s.c., twice daily for 10 days). (B) Leukocyte adhesion values under the same experimental conditions. Data are expressed as the mean ± SEM. Numbers at the base of the bars indicate the number of mice studied in each group. Pharmacologically active doses of gAd prevent abnormal leukocyte–endothelial cell interactions in the microcirculation in the face of chronic adiponectin deficiency.

Figure 2. Endogenous adiponectin deficiency decreases leukocyte rolling velocity in the microcirculation.

Panels illustrate rolling velocities in all experimental groups of mice. Data are expressed as the mean and represent the total leukocyte rolling frequency calculated from 5 mice in each group. Treatment of Ad^–/– mice with human recombinant gAd (25 μg, s.c., twice daily for 10 days) normalizes leukocyte rolling velocity (top, middle panel). Similar results were obtained following functional blockade of E-selectin with the mAb 9A9 in Ad^–/– mice (2.1 mg/kg, i.v.; bottom, right panel).

Figure 3. Pharmacologically active doses of gAd reverse TNF-α–induced leukocyte-endothelium interactions in WT mice.

As shown in A and B, 1 μg/kg TNF-α given 2 hours before the experiment increases leukocyte rolling flux and leukocyte adhesion, respectively, in the microcirculation of WT mice. Two 25 μg doses of gAd given 24 hours and 2.5 hours before intravital microscopy significantly attenuates TNF-α–induced leukocyte rolling and adhesion, confirming the potent antiinflammatory action of pharmacologic doses of gAd also in mice expressing physiologic adiponectin levels. Data are expressed as the mean ± SEM. Numbers at the base of the bars indicate the number of mice studied in each group.

Figure 4. Functional eNOS activity is necessary for the antiinflammatory effect of gAd.

Superfusion of the mouse mesenteric microcirculation with the eNOS inhibitor l-NAME increases leukocyte adhesion in WT mice (white bars). Two 25 μg doses of gAd given 24 hours and 2.5 hours before intravital microscopy failed to prevent leukocyte adhesion in response to l-NAME. Moreover, attenuation of leukocyte adhesion by administration of gAd (25 μg/twice daily for 6 consecutive days) to Ad^–/– mice is acutely hampered by superfusion of the microcirculation with l-NAME (black bars). Data are expressed as the mean ± SEM. Numbers at the base of the bars indicate the number of mice studied in each group.

Figure 5. Adiponectin deficiency increases E-selectin and VCAM-1 expression levels in the vascular endothelium of the mouse microcirculation.

Representative sections of ileal venules (Vs) staining positive for E-selectin are shown in the top 3 photomicrographs. E-selectin staining in all experimental groups of mice was detected by avidin/biotin immunoperoxidase technique seen as brown reaction product (arrows). Original magnification, ×500. Bottom graphs show quantification of the percentage of venules staining positive for E-selectin (left) and VCAM-1 (right). Values are mean ± SEM. Numbers at base of bars indicate the number of mice studied in each group. Five tissue sections were studied in each mouse, and 50 venules were analyzed per section.

Figure 6. E-selectin supports leukocyte adhesion in the Ad^–/– microcirculation.

(A) Leukocyte adhesion in Ad^–/– mice before and after in vivo blockade of E-selectin with the E-selectin function–blocking mAb 9A9 (2.1 mg/kg). Leukocyte adhesion was not affected by systemic administration of control IgG1 (2.1 mg/kg). Leukocyte rolling values in Ad^–/– were slightly increased following administration of mAb 9A9, although they failed to reach statistical significance (B). Values represent the mean ± SEM of rolling or adherent leukocytes. Numbers at the base of the bars indicate the number of mice studied for each group.