Published online before print February 21, 2008, doi: 10.1161/ATVBAHA.108.163634
© 2008 American Heart Association, Inc.
Cell Biology and Signaling |
Adiponectin Protects Against Angiotensin II or Tumor Necrosis Factor 
–Induced Endothelial Cell Monolayer Hyperpermeability
Role of cAMP/PKA Signaling
From the Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine (S.-Q.X., K.M., X.W., L.F., S.D., B.J.G.), and the Department of Molecular Physiology and Biophysics (R.G.S.), Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pa.
Correspondence to Barry J. Goldstein, MD, PhD, Director, Division of Endocrinology, Diabetes and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Room 320 Curtis Building, 1015 Walnut Street, Philadelphia, PA 19107. E-mail Barry.Goldstein{at}jefferson.edu
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Abstract |
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Objective— Angiotensin II (Ang II) and tumor necrosis factor (TNF)-
levels increase endothelial permeability, and we hypothesized that adiponectin suppressed these responses in a cAMP-dependent manner.
Methods and Results— The effect of adiponectin on transendothelial electric resistance (TEER) and diffusion of albumin through human umbilical vein and bovine aortic endothelial cell monolayers induced by Ang II (100 nmol/L) or TNF- (5 ng/mL) was measured. Treatment with the globular domain of adiponectin (3 µg/mL) for 16 hours abrogated the adverse TEER effect of TNF- (–35 versus –12 
/cm2 at 45 minutes, P<0.05) and Ang II (–25 versus –5 /cm2 at 45 minutes, P<0.01) and partially suppressed the increased diffusion of albumin with Ang II (40% versus 10% change, P<0.05) or TNF- (40% versus 20% change, P<0.05). Full-length adiponectin also suppressed Ang II–induced monolayer hyperpermeability. Adiponectin treatment also suppressed Ang II–induced increased actin stress fiber development, intercellular gap formation, and β-tubulin disassembly. Adiponectin increased cAMP levels, and its effects were abrogated by inhibition of adenylyl cyclase or cAMP-dependent protein kinase signaling.
Conclusions— Adiponectin protects the endothelial monolayer from Ang II or TNF--induced hyperpermeability by modulating microtubule and cytoskeleton stability via a cAMP/ PKA signaling cascade.
Full-length and globular adiponectin abrogated the hyperpermeability of an endothelial monolayer elicited by TNF- or Ang II, measured by electric resistance or albumin diffusion. Hyperpermeability correlated with actin stress fiber development, intercellular gap formation, and β-tubulin disassembly, which were all suppressed by adiponectin. Adiponectin increased cellular cAMP, and its effects were blocked by inhibition of cAMP/PKA signaling.
Key Words: adiponectin • angiotensin II • tumor necrosis factor- • permeability • endothelial function • signal transduction • cyclic AMP • protein kinase A
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Introduction |
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The vascular endothelium serves a variety of functions in normal physiology, not the least of which is maintaining the integrity of the barrier function of the endovascular surface.1 In obesity and insulin-resistant states, increases in the levels of several circulating substances, including free fatty acids, angiotensin II (Ang II), cytokines such as tumor necrosis factor (TNF-
), and high glucose in patients with diabetes, exert deleterious effects on endothelial function.2 By various mechanisms, these agents initiate an endothelial inflammatory signaling cascade associated with deficient nitric oxide generation, increased reactive oxygen species generation, increased cell-surface expression of leukocyte adhesion molecules, enhanced leukocyte-endothelial cell adhesion, and alterations in the structural integrity of the cytoskeleton.3 The impaired vascular function and increased permeability associated with these responses contributes to the early stages of the atherogenic process.
Adiponectin, an adipocyte-specific secreted protein, has salutary effects on endothelial function, but in contrast to the substances mentioned above, levels of adiponectin in the circulation are reduced in patients with visceral adiposity.4 A relative deficiency of adiponectin in the bloodstream has been shown to be strongly associated with insulin resistant-disease states as well as coronary artery disease.5,6 Adiponectin exerts several cellular effects that oppose the adverse influences of multiple factors including Ang II, TNF-, and high glucose, which, on balance, determine the degree of impairment of several vascular functional parameters.7 Adiponectin enhances endothelial nitric oxide generation, reduces superoxide production, and suppresses endothelial cell inflammatory signaling and proliferative responses.8
Recent work has begun to decipher the signaling responses to adiponectin at a cellular level. However, the pathway(s) by which adiponectin exerts its salutary effects in vascular endothelial cells remain incompletely defined. 5'-AMP-activated protein kinase (AMP kinase) has clearly been shown to be central to the mechanism of adiponectin signaling in metabolic tissues, including liver, skeletal muscle, and adipocytes, where it facilitates the action of insulin.9 AMP kinase is also at least partly responsible for some of the vascular effects of adiponectin in endothelial cells, including nitric oxide generation.10 In addition, our recent data and work from others has provided evidence that additional pathways are involved in adiponectin endothelial signaling, in particular the cAMP-protein kinase A (PKA) pathway.8,11,12 Both the recombinant C-terminal globular domain of adiponectin (gAd) expressed in bacteria and eukaryotically-expressed full-length adiponectin (fAd) suppress production of reactive oxygen species (ROS) induced by high glucose in endothelial cells primarily via a cAMP-coupled mechanism.12 The effect of increasing cAMP to suppress ROS production was also potentially enhanced by cross talk with the AMP kinase pathway. Overall, however, the cAMP/PKA pathway has emerged as a major signaling system mediating effects of adiponectin in endothelial cells to reverse the adverse cellular effects of TNF- or high glucose.
cAMP and activation of PKA signaling are recognized components of pathways responsible for maintaining endothelial barrier integrity in several vascular beds.1 A number of endothelial responses associated with increased permeability have been shown to be suppressed by the cAMP/PKA pathway including actin stress fiber formation, microtubule disassembly, Rho activation, and myosin light chain phosphorylation leading to intercellular gap formation. In the present study, we tested the hypothesis that adiponectin protects against hyperpermeability of an endothelial cell monolayer induced by Ang II or TNF-. In addition, the potential role of the cAMP/PKA signaling cascade in adiponectin suppression of endothelial permeability was evaluated.
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Methods |
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Materials
Human umbilical vein endothelial cells (HUVECs), endothelial basal medium-2 (EBM-2), and endothelial cell growth supplements were obtained from Lonza. Bovine aortic endothelial cells (BAECs) and BAEC growth medium were obtained from Cell Applications. Endothelial cell serum-free basal medium was from Gibco. The pFLAGCMV3 expression vector and reagents including Ang II and TNF-
and enzyme activators and inhibitors were obtained from Sigma-Aldrich. The transendothelial electric resistance (TEER) measurement system including the EVOMX volt-ohmmeter and the ENDOHM tissue resistance measurement chamber outfitted for a 24-well culture dish insert format was from World Precision Instruments (www.wpiinc.com). Anti–β-tubulin antibody was from Cell Signaling. Fluorescently-labeled secondary antibodies and phalloidin were from Molecular Probes.
Recombinant Globular Domain and Full-Length Adiponectin
The recombinant globular domain of human adiponectin (gAd) was generated in E coli and purified as described.12 Full-length human adiponectin (fAd) was obtained from a eukaryotic expression system (please see http://atvb.ahajournals.org). Protein concentrations were measured using the method of Bradford.13
Cell Culture
HUVECs were cultured in endothelial basal medium-2 containing 10% fetal calf serum (FCS) and premixed endothelial cell growth supplements following the manufacturer’s instructions. BAECs were cultured in BAEC growth medium provided by the manufacturer (Cell Applications). Cells were routinely studied before the fourth passage and at 80% to 90% confluence, except for permeability measurements and cytoskeletal analyses, which required the cells to be fully confluent before experimentation. Before the indicated treatments, the cells were made quiescent by replacing growth medium with serum-free growth medium, containing 5 mmol/L glucose and 1% (wt/vol) bovine serum albumin (BSA) and without growth factor supplementation.
Permeability Measurement Using the TEER System
Permeability measurements were performed by minor modifications of techniques recently published by several groups.14,15 (please see http://atvb.ahajournals.org).
Monolayer Permeability to Albumin
The transmembrane passage of albumin was performed by placing a high concentration of albumin (40 mg/mL) in the lower Transwell chamber and after a 2-hour incubation at 37°C, the diffusion the albumin through the endothelial monolayer barrier was measured in the upper chamber.16 In the control samples, baseline mean diffusion of albumin at 2 hours was 94 µg/mL.
Measurement of Cellular cAMP Content
cAMP was measured in the HCAECs using a direct enzyme immunoassay kit and instructions provided by the manufacturer (GE Healthcare/Amersham Biosciences).
Immunocytochemistry
HUVECs grown on glass coverslips were washed with EBM-2 containing 0.5% (wt/vol) BSA and fixed in 3.7% (wt/vol) formaldehyde solution in phosphatase-buffered saline (PBS) for 10 minutes at 4°C. After washing 3 times with PBS, cells were permeabilized with 0.2% (v/v) Triton X-100 in PBS (PBST) for 30 minutes at room temperature, then blocked with 2% (wt/vol) BSA in PBST for 30 minutes. Incubation with anti–β-tubulin antibody was performed in blocking solution for 1 hour at room temperature followed by staining with Alexa Fluor 594-conjugated secondary antibodies. Actin filaments were stained with Alexa Fluor 594-conjugated phalloidin for 1 hour at room temperature. After immunostaining, the glass slides were analyzed using a phase-contrast inverted fluorescence microscope (Nikon Eclipse TE 2000-U) connected to a digital camera and a video imaging system. For measurement of stress fiber formation, the ratio of the cell area covered by stress fibers to the whole cell area was determined by Journal software version 7.0rl (Metamorph), performed in Kimmel Cancer Center Bioimaging Resource. The values were evaluated statistically with Sigma Plot 8.0 (SPSS Science).
Statistical Analyses
Each set of TEER and albumin permeability transfer experiment were performed in 3 independent experiments, each containing duplicate or triplicate Transwells. Statistical analysis was based on Student t test for comparison of 2 groups or 1-way ANOVA for multiple comparisons. A probability value of <0.05 was used to determine statistical significance.
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Results |
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Adiponectin Ameliorates Endothelial Cell Monolayer Integrity Perturbed by Ang II or TNF-
as Assessed by TEEREndothelial monolayer integrity was assessed by measuring transendothelial electric resistance (TEER) in a 24-well tissue culture plate format. TNF-
and Ang II, 2 well-known agents that increase permeability of the endothelial barrier were tested. The characteristic time-course responses of this system to TNF- (5 ng/mL) or Ang II (100 nmol/L) are shown in Figure 1A and 1B, respectively. Each agent perturbs the integrity of the HUVEC monolayer leading to a significant decrease in electric resistance of –35 or –25 /cm2 membrane surface area at the end of the 45-minute incubation period with TNF- or Ang II, respectively. Under these conditions, prior incubation with gAd (3 µg/mL) for 16 hours significantly abrogated the effect of both TNF- and Ang II, decreasing the net permeability-inducing effect of TNF- to –12 /cm2 at 45 minutes (P<0.05) and that of Ang II to –5 /cm2 at 45 minutes (P<0.01). Inclusion of the endothelial nitric oxide synthase (eNOS) inhibitor N
-nitro-L-arginine methyl ester (L-NAME) at 10 µmol/L for 30 minutes before and during measurement of TEER for 45 minutes had no effect on basal resistance or on adiponectin suppression of increased permeability induced by either TNF- or Ang II (data not shown). Thus, the effect of adiponectin to suppress the increased permeability induced by these agonists does not involve the known ability of adiponectin to increase activity of eNOS.
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To test another endothelial cell type to compare with HUVECs, we also performed TEER analysis using BAECs and treating them similarly with TNF- (5 ng/mL) or Ang II (100 nmol/L) as well as evaluating the effect of a 16-hour prior incubation with gAd (3 µg/mL). The results at 15 and 45 minutes postagonist treatment were similar. The baseline mean TEER value among all of the samples was 27.4 /cm2. Compared to control, at 45 minutes TNF- and Ang II elicited 32.2±3.0 and 23.2±1.7 /cm2 decreases in the measured TEER values, respectively (n=3, both P<0.05). Treatment with gAd completely abrogated the reduction in TEER measured after TNF- to +3.0±4.6 from baseline, and significantly reduced the change in TEER from baseline after Ang II to only –7.7±1.2. Thus, gAd exhibited a similar protective effect against increased permeability induced by TNF- and Ang II in endothelial cells from different anatomic sources.
Adiponectin Ameliorates Endothelial Cell Monolayer Integrity Perturbed by Ang II or TNF- as Assessed by Albumin Permeability
To complement the TEER system, which measures ion conductance through the endothelial monolayer, parallel experiments were also performed using a method that measure the transmembrane movement of albumin. HUVECs were cultured on 0.45-µm membrane pore size culture inserts precoated with fibronectin (supplemental Figure I, available online at http://atvb.ahajournals.org). After adding 40 mg/mL bovine serum albumin to the lower chamber, the cells were then treated with either Ang II (100 nmol/L) or TNF- (5 ng/mL) for 2 hours, and the upper chamber was sampled for measurement of albumin by protein assay.16 During the 2-hour incubation, Ang II and TNF- elicited similar increases in albumin transfer by 40% over the control level. Preincubation with 3 µg/mL gAd for 16 hours before the addition of Ang II or TNF- and 40 mg/mL bovine serum albumin to the lower chamber, significantly reduced the effect of Ang II and TNF- to increase transmonolayer diffusion of albumin. The response to gAd was somewhat more pronounced with Ang II (40% versus 10% change, P<0.05) compared to TNF- (40% versus 20% change, P<0.05).
Dose-Response of Globular and Full-Length Adiponectin on Suppression of HUVEC Monolayer Permeability Induced by Ang II
The dose-response of the recombinant adiponectin globular domain (gAd) as well as full-length adiponectin (fAd) purified from a eukaryotic expression system was tested in the suppression of HUVEC monolayer permeability induced by Ang II (Figure 2). Although a 4-hour incubation with either of the 2 adiponectin isoforms had minimal effects on the basal level of monolayer permeability, both gAd and fAd exhibited a similar dose-response for reducing the effect of a 45-minute treatment with 100 nmol/L Ang II. Ang II alone induced a net increase of permeability by 27 /cm2. Pretreatment with increasing concentrations of gAd or fAd (1, 3, or 6 µg/mL) for 4 hours significantly abolished the Ang II–induced hyperpermeability in a dose-dependent manner and to a similar extent compared to control cells not treated with Ang II.
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The Effect of Adiponectin to Ameliorate Ang II–Induced Monolayer Permeability Is Mediated by cAMP/PKA Signaling
We next carried out experiments to determine whether the known effect of adiponectin to increase endothelial cAMP and stimulate PKA activity was involved in the protective effect of adiponectin on Ang II–induced hyperpermeability (Figure 3). In these experiments, treatment with 100 nmol/L Ang II (45 minutes) induced a net increase of permeability of 22.5 /cm2. Pretreatment with gAd for 4 hours nearly abolished the effect of Ang II (Figure 3). This response was similar to the effect of treatment with the adenylyl cyclase activator forskolin (2 µmol/L) for 45 minutes, which increased the transendothelial resistance of the HUVEC monolayer and abolished the Ang II–induced hyperpermeability (supplemental Figure II). As a cAMP mimetic, the cell-permeable analogue dibutyryl-cAMP (db-cAMP, 5 or 50 µmol/L) increased monolayer resistance measured by TEER above baseline, suggesting enhanced integrity of the diffusion barrier (supplemental Figure III). Prior treatment of the monolayer with 1 µmol/L db-cAMP completely prevented the exacerbation of monolayer permeability induced by Ang II (supplemental Figure IV). These data support the hypothesis that cAMP/PKA signaling is a major mechanism that protects against Ang II–induced endothelial permeability.
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To test the potential role of cAMP/PKA signaling in the action of adiponectin on Ang II–mediated endothelial permeability, treatment with the adenylyl cyclase inhibitor, dd-Ado (100 µmol/L) or the PKA inhibitor Rp-cAMP (10 µmol/L) 30 minutes before incubation blocked the protective effect of either gAd (Figure 3) or fAd (supplemental Figure II) on Ang II–induced reduction in TEER. These data suggest that the protective effect of adiponectin against Ang II–induced hyperpermeability is substantially mediated via the cAMP/PKA signaling transduction pathway.
Time Course of Adiponectin Induction of Increased cAMP Levels in HUVECs
Because the salutary effects of adiponectin on endothelial monolayer permeability were blocked by inhibitors of cAMP/PKA signaling, we further demonstrated that adiponectin increased cAMP levels in HUVECs (supplemental Figure V). Over a time course corresponding to the Transwell experiments shown above, the basal levels of cellular cAMP content (2.57±0.25 pmoles/µg protein) increased by less than 10% (P>0.05) after a 15-minute treatment with 3 µg/mL gAd. However, by 45 minutes, cAMP levels were significantly increased by 38% to 3.65±0.38 pmoles/µg protein (P<0.005) and continued to steadily rise to a level of 88% above basal at 16-hour incubation (5.80±0.49 pmoles/µg protein; P<0.001). This result supports the cAMP/PKA pathway in the mechanism of adiponectin action in endothelial cells.
Adiponectin Suppression of Ang II– or Nocodazole-Induced Actin Reorganization and Microtubule Disruption
To evaluate some of the underlying structural alterations accompanying the cytoskeletal elements and microtubule assembly that determines the integrity of cell-cell contacts, we extended our observations to the abundance of F-actin and β-tubulin in response to the various agents tested above. HUVECs were grown on glass coverslips, incubated in reduced serum medium, and preincubated as indicated with either gAd or fAd (3 µg/mL, 3 hour), dd-Ado (100 µmol/L), or Rp-cAMP (10 µmol/L) for 30 minutes before stimulation with Ang II or nocodazole (Figure 4). Stimulation with either Ang II (100 nmol/L) or nocodazole (200 nmol/L), a pharmacological agent that disrupts microtubules, dissolved the cortical actin filaments and triggered stress fiber accumulation as evidenced by parallel actin filaments and paracellular gap formation (Figure 4). Quantitative analysis of F-actin abundance revealed that adiponectin pretreatment significantly attenuates Ang II–induced stress fiber formation (supplemental Figure VI; 21% of actin stress fiber area per cell in Ang II–treated cells compared with 15% of actin stress fiber area per cell in adiponectin-pretreated cells followed by Ang II stimulation, P<0.05). Consistent with the TEER measurements discussed above, preincubation with gAd or fAd abolished Ang II– and nocodazole-induced stress fiber and gap formation. In addition, the inhibitors dd-Ado or Rp-cAMP blocked the protective effect of gAd (P<0.001), consistent with a role for cAMP signaling in the effects of adiponectin on the Ang II–induced microskeletal alterations. These results strongly suggest the involvement of actin rearrangement in protective effect of intracellular cAMP elevation and PKA activation on Ang II–induced cytoskeletal remodeling and barrier compromise.
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Immunofluorescent analysis of intracellular microtubule arrangements with an anti–β-tubulin antibody shows that adiponectin alone did not affect microtubule structure in HUVECs (Figure 5). Endothelial cell pretreatment with adiponectin before Ang II stimulation prevented microtubule disassembly induced by Ang II (Figure 5E and 5F). The inhibitors dd-Ado and Rp-cAMP abrogated the protective effect of adiponectin on Ang II–induced microtubule disassembly in HUVEC (Figure 5H, 5I, 5K, and 5L).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study demonstrates that adiponectin protects against increased permeability of an endothelial cell monolayer induced by TNF-
or Ang II in a cAMP/PKA-dependent manner. Adiponectin suppressed multiple cellular effects known to be associated with endothelial hyperpermeability including actin stress fiber development, intercellular gap formation, and β-tubulin disassembly. The full-length form of adiponectin and the recombinant globular domain both protected the integrity of the endothelial barrier. While this work was being prepared for submission, Elbatarny et al17 reported that adiponectin increased the integrity of a microvascular endothelial cell monolayer to the passage of fluorescein-conjugated dextran; however, the signaling mechanism of adiponectin was not evaluated.
These results are consistent with in vivo measures of endothelial function in several studies showing endothelium-dependent forearm vasodilation in humans negatively correlated with adiponectin.18–21 as well as a reduction in endothelium-dependent vasodilation in aortic rings from adiponectin knock-out mice.18 We recently showed in vivo using intravital microscopy in adiponectin knock-out mice that adiponectin deficiency is associated with an inflammatory phenotype leading to a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion to the venular microcirculation.22 Deficiency of adiponectin elicited an inflammatory vascular phenotype with increased expression of E-selectin and vascular cell adhesion molecule (VCAM)-1 that was reversed with systemic administration of the recombinant globular fragment of adiponectin. In cultured cells, adiponectin has been shown to reverse the deleterious effects of TNF- and other cytokines on endothelial function. TNF--induced expression of several adhesion molecules, including VCAM-1, E-selectin, and intercellular adhesion molecule-1 (ICAM-1), was blocked by adiponectin.23,24 Endothelial Ang II–mediated effects leading to reduced nitric oxide availability can be reversed by adiponectin, which restores coupling between eNOS and HSP90.25 Interestingly, in the present work, we found that the effect of adiponectin to suppress the increased permeability induced by Ang II and TNF- was not affected by inhibition of eNOS, suggesting an alternative pathway for the protective effect of adiponectin on endothelial monolayer permeability.
Work by several groups over the past decade has helped elucidate the multiple signaling mechanisms affecting the cortical actin cytoskeletal network and cellular contractile events that tether intercellular adhesive structures and disrupt the integrity of the endothelial barrier leading to the passage of fluid, ions, and proteins.1 The major effects leading to cellular structural changes eventually result in phosphorylation of myosin light chain (MLC) by MLC kinase as well as Rho-dependent mechanisms in a variety of endothelial cell types.26,27 Rho-kinase can directly phosphorylate MLC and inactivate the myosin-associated protein phosphatase type 1.28 Upstream of these events, a major role has been attributed to cAMP and PKA activation in the protection of the integrity of the endothelial barrier.29,30 PKA has multiple effects, including phosphorylation of the actin-binding proteins filamin and adductin,31,32 the focal adhesion proteins paxillin and focal adhesion kinase, and it reduces the activity of MLC kinase leading to decreased basal MLC phosphorylation.33 Ultimately, PKA triggers the disappearance of stress fibers and filamentous actin (F-actin) accumulation in membrane ruffles34 and blocks microtubule disassembly effectively protecting the endothelial barrier integrity.28,35,36
PKA also modulates Rho GTPase activity resulting in inhibition of RhoA activity and stabilization of cortical actin cytoskeleton, which promotes endothelial barrier enhancement.37,38 Interestingly, in separate work examining vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells, we have recently found that RhoA activation is suppressed by adiponectin in a cAMP/PKA dependent manner.39 This effect also supports a major role for cAMP signaling in the overall effects of adiponectin in endothelial cells.
Adiponectin elicits salutary responses in endothelial cells that oppose diverse adverse influences, including the effects of Ang II and TNF-, which are known to trigger different cellular signaling pathways. This observation suggests that adiponectin exerts a common protective effect in the endothelium. Ang II affects the endothelial barrier function through its AT1 receptor, which increases fluid leak by stimulating both cGMP synthesis and cAMP degradation.40 TNF- activates sphingomyelinase leading to generation of ceramide with subsequent activation of a number of signaling kinases, increased cellular ROS generation, and reduced nitric oxide availability.41–43 TNF- induces cytoskeletal rearrangement by phosphorylation of myosin light chain (MLC) kinase, ultimately leading to cell shape changes and the formation of paracellular gaps.44,45 TNF--induced endothelial permeability has also been linked to cAMP signaling. TNF- can reduce endothelial cAMP levels via increased phosphodiesterase 2A expression and activity via upstream activation of MAPK p38.46
A role for cAMP signaling in adiponectin action in endothelial cells has been strongly implicated in other recent studies. We reported that both recombinant bacterial gAd and eukaryotically-expressed fAd suppress ROS production induced by high glucose in endothelial cells.12 Globular adiponectin increased cellular cAMP content, inhibition of PKA blocked the effect of either gAd or fAd to suppress ROS generation, and the suppression of glucose-induced ROS was also mimicked by increasing cellular cAMP by treatment with forskolin, which activates adenylyl cyclase or using the cAMP mimetic, dibutyryl cAMP, indicating that adiponectin suppression of ROS production in high glucose is mediated by a cAMP/PKA dependent-pathway. Ouchi et al11 also showed that gAd suppression of TNF--induced I
B phosphorylation and NFB activation in endothelial cells was accompanied by cAMP accumulation and was blocked by inhibitors of adenylyl cyclase or PKA. In murine peritoneal macrophages, gAd was recently shown to increase cAMP and PKA activity and reduce leptin-induced ERK1/2 and p38 MAPK phosphorylation.46 Together, these findings help establish the cAMP/PKA pathway as a major signaling system mediating effects of adiponectin in endothelial cells. Further work will help define the protective signaling mechanism in the endothelium by which adiponectin increases cAMP levels.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants DK63018 and DK71360 to Dr Goldstein.
Disclosures
None.
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Footnotes |
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S.-Q.X. and K.M. contributed equally to this study.
Original received July 30, 2007; final version accepted February 12, 2008.
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