James Surapisitchat

Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1 stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. TNF-α and IL-1 regulate gene expression in ECs, in part, by stimulating mitogen-activated protein kinases (MAPK), which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAPK in EC. To test this hypothesis, we determined the effects of flow (shear stress = 12 dynes/cm2) on TNF-α and IL-1-stimulated activity of three MAPK in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 and p38 activity but decreased JNK activity compared with static controls. TNF-α or IL-1 alone activated ERK1/2, p38, and JNK maximally at 15 min in HUVEC. Preexposing HUVEC for 10 min to flow inhibited TNF-α and IL-1 activation of JNK by 46% and 49%, respectively, but had no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, which inhibits flow-mediated ERK1/2 activation, prevented flow from inhibiting cytokine activation of JNK. Phorbol 12-myristate 13-acetate, which strongly activates ERK1/2, also inhibited TNF-α activation of JNK. These findings indicate that fluid shear stress inhibits TNF-α-mediated signaling events in HUVEC via the activation of the ERK1/2 signaling pathway. Inhibition of TNF-α signal transduction represents a mechanism by which steady laminar flow may exert atheroprotective effects on the endothelium.

Endothelial atheroprotective and anti-inflammatory mechanisms

Abstract: Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor‐α (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF‐mediated EC activation. EC apoptosis is likely important in the process termed “plaque erosion” that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide‐dependent protective pathways that involve enzymes PI3‐kinase and Akt. Conversely, our laboratory has identified nitric oxide‐independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen‐activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF‐mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF‐stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c‐Jun N‐terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre‐exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen‐activated protein kinase kinase inhibitor, blocked the flow‐mediated inhibition of TNF activation of JNK. Flow‐mediated inhibition of JNK was unaffected by 0.1 mM L‐nitroarginine, 100 μM 8‐bromo‐cyclic GMP, or 100 μM 8‐bromo‐cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow‐mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Angiotensin II Stimulates p21-Activated Kinase in Vascular Smooth Muscle Cells: Role in Activation of JNK

Angiotensin II (Ang II) has been previously shown to stimulate the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase family members. Little is known regarding the upstream signaling molecules involved in Ang II-mediated JNK activation. Ang II has been shown to activate the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway, suggesting similarities to cytokine signaling. In response to cytokines such as interleukin-1 and tumor necrosis factor-alpha, the p21-activated kinase (PAK) has been identified as an upstream component in JNK activation. Therefore, we hypothesized that PAK may be involved in JNK activation by Ang II in vascular smooth muscle cells (VSMCs). AlphaPAK activity was measured by myelin basic protein phosphorylation in rat aortic VSMCs. In response to Ang II, alphaPAK was rapidly stimulated within 1 minute, with a peak (5-fold increase) at 30 minutes. AlphaPAK stimulation preceded activation of JNK in VSMCs. Ang II-mediated activation of both alphaPAK and JNK was Ca2+ dependent and inhibited by downregulation of phorbol ester-sensitive protein kinase C isoforms (by pretreatment with phorbol 12,13-dibutyrate) but not by pretreatment with GF109203X. Activation of both PAK and JNK was partially inhibited by tyrosine kinase inhibitors but not by specific Src inhibitors, suggesting regulation by a tyrosine kinase other than c-Src. Finally, introduction of dominant negative PAK markedly reduced the JNK activation by Ang II in both Chinese hamster ovary and COS cells stably expressing the Ang II type 1 receptor (AT1R). Our data provide evidence for alphaPAK as an upstream mediator of JNK in Ang II signaling and extend the role of Ang II as a proinflammatory mediator for VSMCs.

Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3

The inflammatory cytokine TNF-alpha stimulates several presumed pro-atherogenic signaling events in endothelial cells (ECs), including activation of c-Jun NH(2)-terminal kinase (JNK) and induction of E-selectin. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, is required for TNF-mediated JNK activation. TNF activates ASK1 in part by dissociating ASK1 from its inhibitor 14-3-3. Because the risk of atherosclerosis is decreased in regions of steady laminar flow, we hypothesized that laminar flow inhibits proinflammatory cytokine-mediated activation of JNK. Steady laminar flow inhibited both TNF activation of ASK1 and JNK. Inhibition of ASK1 by flow correlated with increased association of ASK1 with 14-3-3. A constitutively active form of ASK1 lacking the 14-3-3-binding site (ASK1-Delta NS967A) was not inhibited by flow. These data establish ASK1 as a target for flow-mediated inhibition of cytokine signaling and indicate a novel role for 14-3-3 as an anti-inflammatory mediator in ECs.

Differential regulation of endothelial cell permeability by cGMP via phosphodiesterases 2 and 3

Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-(O-[N-methylpiperazino]-&ggr;-butyryl)-dihydrochloride–forskolin (MPB–forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB–forskolin on thrombin-induced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-&agr; reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-&agr; on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells.

Fluid shear stress inhibits TNF-mediated JNK activation via MEK5-BMK1 in endothelial cells

Steady laminar blood flow protects vessels from atherosclerosis. We showed that flow decreased tumor necrosis factor-alpha (TNF)-mediated VCAM1 expression in endothelial cells (EC) by inhibiting JNK. Here, we determined the relative roles of MEK1, MEK5 and their downstream kinases ERK1/2 and BMK1 (ERK5) in flow-mediated inhibition of JNK activation. Steady laminar flow (shear stress=12dyn/cm(2)) increased BMK1 and ERK1/2 activity in EC. Pre-exposing EC for 10min to flow inhibited TNF activation of JNK by 58%. A key role for BMK1, but not ERK1/2 was shown. (1) Incubation of EC with PD184352, at concentrations that blocked ERK1/2, but not BMK1, had no effect on flow inhibition of TNF-mediated JNK activation. (2) BIX02188, a MEK5-selective inhibitor, completely reversed the inhibitory effects of flow. These findings indicate that flow inhibits TNF-mediated signaling events in EC by a mechanism dependent on activation of MEK5-BMK1, but not MEK1-ERK1/2. These results support a key role for the MEK5-BMK1 signaling pathway in the atheroprotective effects of blood flow.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Regulation of Endothelial Barrier Function by Cyclic Nucleotides: The Role of Phosphodiesterases

The endothelium plays an important role in maintaining normal vascular function. Endothelial barrier dysfunction leading to increased permeability and vascular leakage is associated with several pathological conditions such as edema and sepsis. Thus, the development of drugs that improve endothelial barrier function is an active area of research. In this chapter, the current knowledge concerning the signaling pathways regulating endothelial barrier function is discussed with a focus on cyclic nucleotide second messengers (cAMP and cGMP) and cyclic nucleotide phosphodiesterases (PDEs). Both cAMP and cGMP have been shown to have differential effects on endothelial permeability in part due to the various effector molecules, crosstalk, and compartmentalization of cyclic nucleotide signaling. PDEs, by controlling the amplitude, duration, and localization of cyclic nucleotides, have been shown to play a critical role in regulating endothelial barrier function. Thus, PDEs are attractive drug targets for the treatment of disease states involving endothelial barrier dysfunction.

Chapter 173 – Phosphodiesterase Families

Publisher Summary There are 11 known phosphodiesterase (PDE) gene families, each with their own distinct characteristics. The phosphodiesterases share the same organizational structure. Each protein has an N-terminal domain that confers regulatory properties to the protein, followed by a more C-terminal ∼270 amino acid catalytic domain and a short C-terminal tail. The sequence identity in the catalytic domain between genes is only about 35 percent, yet all PDEs possess the signature sequence H-D-X2-H-X4-N. The substrate specificities of the different PDE families run the gamut from dual-specificity PDEs to those that are highly specific for either cAMP or cGMP. The PDE1 proteins have two Ca2+/calmodulin binding domains, and binding of calmodulin to these PDEs stimulates their activity. The PDE2, PDE5, PDE6, PDE10, and PDE11 proteins all have allosteric, cyclic nucleotide-binding domains known to be part of the larger GAF domain family. The PDE4 family, a large family of enzymes with four genes and many splice variants, is responsible for the majority of basal cAMP-hydrolyzing activity in many cell types. Different PDEs are subject to protein phosphorylation by a variety of kinases that can alter PDE activity. In pancreatic β cells, PDE3B can be phosphorylated and activated by PKB in response to leptin stimulation. Various members of the PDE4 family can be phosphorylated and regulated by PKA and ERK. PDE5 can be phosphorylated by PKG, stabilizing it ability to bind and be activated by cGMP. The PDE superfamily comprises a complex set of enzymes that can provide cross-talk between the cGMP and cAMP pathways, and with Ca2+/CaM-dependent pathways and various kinase pathways, and allow the cell exquisite control of cyclic nucleotide dynamics.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.