Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB.
To elucidate the mechanisms regulating matrix-specific PAK activation.
Methods and Results
We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI.
Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK.
Shear stress; extracellular matrix; protein kinase A; p21 activated kinase; NF-κB
Atherosclerosis begins as local inflammation of artery walls at sites of disturbed flow. The c-Jun NH2-terminal kinase (JNK) is thought to be one of the major regulators of flow-dependent inflammatory gene expression in endothelial cells in atherosclerosis. We now show that JNK activation by both onset of laminar flow and long-term oscillatory flow is matrix-specific, with enhanced activation on fibronectin compared to basement membrane protein or collagen. Flow-induced JNK activation on fibronectin requires new integrin ligation, and requires both the MAP kinase kinase MKK4 and p21-activated kinase (PAK). In vivo, JNK activation at sites of early atherogenesis correlates with the deposition of fibronectin. Inhibiting PAK reduces JNK activation in atheroprone regions of the vasculature in vivo. These results identify JNK as a matrix-specific, flow-activated inflammatory event. Together with other studies, these data elucidate a network of matrix-specific pathways that determine inflammatory events in response to fluid shear stress.
shear stress; atherosclerosis; JNK
PAK2 mediates shear stress–induced NF-κB activation. Basement membrane proteins limit the proinflammatory response to shear by blocking the interaction of PAK2 with the adaptor protein Nck. This uncoupling response requires protein kinase A–dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain.
Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A–dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A–dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-κB phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow–induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide–dependent NF-κB inhibition.
Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability. We now report that onset of fluid flow, atherogenic flow profiles, oxidized LDL, and proatherosclerotic cytokines all stimulate PAK phosphorylation and recruitment to cell–cell junctions. Activation of PAK is higher in cells plated on fibronectin (FN) compared to basement membrane proteins in all cases. In vivo, PAK is activated in atherosclerosis-prone regions of arteries and correlates with FN in the subendothelium. Inhibiting PAK in vivo reduces permeability in atherosclerosis-prone regions. Matrix-specific PAK activation therefore mediates elevated vascular permeability in atherogenesis.
PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on in vitro studies and its role in angiogenesis in vivo has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1−/− and PAK1+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1−/− but not PAK1+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1−/− and PAK1+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.
Bone morphogenetic proteins (Bmps) are important mediators of inflammation and atherosclerosis, though their mechanism of action is not fully understood. To better understand the contribution of the Bmp signaling pathway plays in vascular inflammation, we investigated the role of Bmper (Bmp-endothelial cell precursor-derived regulator), an extracellular Bmp modulator, in an induced in vivo model of inflammation and atherosclerosis.
Methods and Results
We crossed apolipoprotein E-deficient (ApoE−/−) mice with mice missing one allele of Bmper (Bmper+/−, used in place of Bmper−/− mice that die at birth) and measured the development of atherosclerosis in mice fed a high fat diet. Bmper haploinsufficiency in ApoE−/− mice (Bmper+/−;ApoE−/− mice) led to a more severe phenotype compared to Bmper+/+;ApoE−/− mice. Bmper+/−;ApoE−/− mice also exhibited increased Bmp activity in endothelial cells in both the greater and lesser curvatures of the aortic arch, suggesting a role for Bmper in regulating Bmp-mediated inflammation associated with laminar and oscillatory shear stress. siRNA knockdown of Bmper in human umbilical vein endothelial cells caused a dramatic increase in the inflammatory markers ICAM1 and VCAM1 at rest and following exposure to oscillatory and laminar shear stress.
We conclude that Bmper is a critical regulator of Bmp-mediated vascular inflammation and that the fine-tuning of Bmp and Bmper levels is essential in the maintenance of normal vascular homeostasis.
Bone morphogenetic protein; Bmp endothelial cell precursor-derived regulator; atherosclerosis; inflammation; fluid shear stress
Disturbed blood flow promotes atherosclerosis mainly by inducing inflammatory gene expression in endothelial cells. Flow stimulates the proinflammatory transcription factor NF-κB through integrin- and Rac-dependent production of reactive oxygen species (ROS). Previous work demonstrated that NF-κB activation by flow is matrix-specific, occurring in cells on fibronectin but not collagen. Activation of p21-activated kinase (PAK) followed the same matrix-dependent pattern. We now show that inhibiting PAK in cells on fibronectin blocked NF-κB activation by both laminar and oscillatory flow in vitro and at sites of disturbed flow in vivo. Constitutively active PAK rescued flow-induced NF-κB activation in cells on collagen. Surprisingly, PAK was not required for flow-induced ROS production. Instead, PAK modulated the ability of H2O2 to activate the NF-κB pathway. These data demonstrate that PAK controls NF-κB activation by modulating cells’ sensitivity to ROS.
Atherosclerosis begins as local inflammation of arterial walls at sites of disturbed flow, such as vessel curvatures and bifurcations with low shear stress. c-Jun NH2-terminal kinase (JNK) is a major regulator of flow-dependent gene expression in endothelial cells in atherosclerosis. However, little is known about the in vivo role of JNK in low shear stress in atherosclerosis. We aimed to observe the effect of JNK on low shear stress–induced atherogenesis in apolipoprotein E-deficient (ApoE−/−) mice and investigate the potential mechanism in human umbilical vein endothelial cells (HUVECs). We divided 84 male ApoE−/− mice into two groups for treatment with normal saline (NS) (n = 42) and JNK inhibitor SP600125 (JNK-I) (n = 42). Perivascular shear stress modifiers were placed around the right carotid arteries, and plaque formation was studied at low shear stress regions. The left carotid arteries without modifiers represented undisturbed shear stress as a control. The NS group showed atherosclerotic lesions in arterial regions with low shear stress, whereas the JNK-I group showed almost no atherosclerotic lesions. Corresponding to the expression of proatherogenic vascular cell adhesion molecule 1 (VCAM-1), phospho-JNK (p-JNK) level was higher in low shear stress regions with NS than with JNK-I inhibitor. In HUVECs under low shear stress, siRNA knockdown and SP600125 inhibition of JNK attenuated nuclear factor (NF)-κB activity and VCAM-1 expression. Furthermore, siRNA knockdown of platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31) reduced p-JNK and VCAM-1 levels after low shear stress stimulation. JNK may play a critical role in low shear stress–induced atherogenesis by a PECAM-1–dependent mechanosensory pathway and modulating NF-κB activity and VCAM-1 expression.
Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis.
Methods and Results
We crossed C57BL/6 Pecam1−/− mice with apolipoprotein E–deficient (Apoe−/−) mice. On a Western diet, Pecam1−/−Apoe−/− mice showed reduced atherosclerotic lesion size compared to Apoe−/− mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-κB were all reduced in Pecam1−/−Apoe−/− mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-κB activity and VCAM-1 expression under conditions of atheroprone flow.
These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-κB–mediated gene expression.
atherosclerosis; shear stress sensing; adhesion molecules; endothelium; macrophages
Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. While many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator which antagonizes cardiac hypertrophy.
Methods and Results
Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs), or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an anti-hypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1cko). When subject to 2 weeks of pressure overload, Pak1cko mice compared to controls, developed greater cardiac hypertrophy with attendant blunting of JNK activation, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. In addition, chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1cko mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analogue, was able to prevent pressure overload-induced hypertrophy in wild-type mice, without compromising their cardiac functions. Meanwhile FTY720 failed to exert such an effect on Pak1cko mice, suggesting that the anti-hypertrophic effect of FTY720 likely acts through Pak1 activation.
These results, for the first time, establish Pak1 as a novel anti-hypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.
Cardiac hypertrophy; heart failure; signal transduction; stress
The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics.
We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src–transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
Increased permeability of blood vessels is an important component of inflammation, but in some circumstances it contributes to tissue injury and organ failure. Previous work showed that p21-activated kinase (PAK) is a critical regulator of endothelial cell–cell junctions through effects on myosin light chain phosphorylation and cell contractility. We now show that blocking PAK function inhibits fluid leak in a mouse model of acute lung injury. In cultured endothelial cells, induction of myosin light chain phosphorylation by PAK is mediated by mitogen-activated protein kinase kinase and extracellular signal-regulated kinase (Erk). Erk in lipopolysaccharide (LPS)-treated mouse lung is activated in a PAK-dependent manner in several cell types, most prominently vascular endothelium. Activation of Erk requires the integrity of the complex between PAK, PIX, and GIT1. Several means of disrupting this complex inhibit stimulation of vascular permeability in vitro. A cell-permeant peptide that blocks binding of PAK to PIX inhibits LPS-induced fluid leak in the mouse lung injury model. We conclude that the PAK–PIX–GIT1 complex is critical for Erk-dependent myosin phosphorylation and vascular permeability.
Earlier investigations in our lab indicated an anti-adrenergic effect induced by activation of p21-activated kinase (Pak-1) and protein phosphatase 2A (PP2A).
Our objective was to test the hypothesis that Pak-1/PP2A is a signaling cascade controlling stress-induced cardiac growth. We determined the effects of ablation of the Pak-1 gene on the response of the myocardium to chronic stress of isoproterenol (ISO) administration.
Methods and Results
Wild-type (WT) and Pak-1-knockout (Pak-1-KO) mice were randomized into six groups to receive either ISO, saline (CTRL), or ISO and FR180204, a selective inhibitor of Erk1/2. Echocardiography revealed that hearts of the Pak-1-KO/ISO group had increased LV fractional shortening, reduced LV chamber volume in diastole and systole, increased cardiac hypertrophy, and enhanced transmitral early filling deceleration time, compared to all other groups. The changes were associated with an increase in relative Erk1/2 activation in Pak-1-KO/ISO mice versus all other groups. ISO-induced cardiac hypertrophy and Erk1/2 activation in Pak-1-KO/ISO were attenuated when the selective Erk1/2 inhibitor FR180204 was administered. Immunoprecipitation showed an association between Pak-1, PP2A, and Erk1/2. Cardiac myocytes infected with an adenoviral vector expressing constitutively active Pak-1 showed a repression of Erk1/2 activation. p38 MAPK phosphorylation was decreased in Pak-1-KO/ISO and Pak-1-KO/CTRL mice compared to WT. Levels of phosphorylated PP2A were increased in ISO-treated Pak-1-KO mice, indicating reduced phosphatase activity. Maximum Ca2+-activated tension in detergent-extracted bundles of papillary fibers from ISO-treated Pak-1-KO mice was higher than in all other groups. Analysis of cTnI phosphorylation indicated that compared to WT, ISO-induced phosphorylation of cTnI was blunted in Pak-1-KO mice.
Active Pak-1 is a natural inhibitor of Erk1/2 and a novel anti-hypertrophic signaling molecule upstream of PP2A.
Mitogen-Activated Protein Kinases; Myocardial hypertrophy; p38 MAPK; Pak-1; Troponin I
The p21-activated serine/threonine protein kinase Pak2/γ-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.
The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.
Rac; Cdc42; cell motility; cytoskeleton; contractility
Gastrins, including amidated gastrin17 and glycine‐extended gastrin17, are important growth factors in colorectal cancer (CRC). The p21‐activated kinase 1 (PAK1) plays key roles in cellular processes including proliferation, survival, and motility, and in cell transformation and tumor progression. PAK1 expression increases with the progression of CRC, and knockdown of PAK1 blocks CRC cell growth and metastasis both in vitro and in vivo. The aim of this study was to determine the interaction between PAK1 and gastrins in CRC cells. PAK1 expression and activation were assayed by Western blots, and concentrations of gastrin mRNA and peptides by real‐time PCR and radioimmunoassay, respectively. Proliferation of CRC cells was measured by 3H‐thymidine incorporation, and vascular endothelial growth factor (VEGF) secretion was measured by ELISA. Gastrins activated PAK1 via PI3K‐dependent pathways. Activated PAK1 in turn mediated gastrin‐stimulated activation of β‐catenin and VEGF secretion in CRC cells, as knockdown of PAK1 blocked stimulation of these cellular processes by gastrins. Downregulation of gastrin reduced the expression and activity of PAK1, but in contrast there was a compensatory increase in gastrins either when PAK1 was downregulated, or after treatment with a PAK inhibitor. Our results indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.
The results in this study indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.
Colon cancer; Gamide; glycine extended; p21‐activated kinase 1
Gliomas display anoikis resistance, enhanced invasion in to the adjacent brain parenchyma and eventually recur despite using the standard therapies. Our studies on increased anoikis sensitization in matrix metalloproteinase-2 (MMP-2)-knockdown 4910 and 5310 human glioma xenograft cells were interestingly correlated with p21-activated kinase 4 (PAK4) inhibition, prompting us to further investigate the role of PAK4 in glioma. Here, we report the PAK4 upregulation in positive correlation with increasing glioma pathological grades. The siRNA-mediated PAK4 knockdown elevated anoikis, and inhibited invasion and migration by downregulating MMP-2, αvβ3-integrin and phospho-epidermal growth factor receptor (phospho-EGFR). The cDNA-PCR arrays revealed a transcriptional suppression of essential proteins involved in cell proliferation and adhesion in PAK4-knockdown cells. Most importantly, glutathione S-transferase pull-down assays demonstrated the MMP-2 as a new PAK4-interacting protein which binds to PAK4 kinase domain. Individual EGFR/ErbB2 inhibitor and αvβ3 antibody treatments in PAK4si-treated cells indicated the regulation of αvβ3/EGFR survival signaling by PAK4. Overexpression of PAK4 significantly reversed the MMP2si-induced cell death in both cell lines. Codepletion of PAK4 and MMP-2 resulted in robust anoikis-mediated cell death, and severely inhibited invasive and migratory properties in these cells. PAK4si inhibited in vivo tumor growth in nude mice by inhibiting MMP-2, β3-integrin and phospho-EGFR levels in tumors. Our findings indicate a physical association between PAK4 and MMP-2, and suggest the future therapeutic potential of PAK4/MMP-2 dual targeting in glioma treatment.
anoikis; αvβ3 integrin; EGFR; MMP-2; PAK4; kinase domain
Aberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR−/− mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown.
The preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS.
These translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis.
The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.
Enhancing structural and functional integrity of mitochondria is an emerging therapeutic option against endothelial dysfunction. In this study, we sought to investigate the effect of fluid shear stress on mitochondrial biogenesis and mitochondrial respiratory function in endothelial cells (ECs) using in vitro and in vivo complementary studies.
Methods and Results
Human aortic- or umbilical vein-derived ECs were exposed to laminar shear stress (20 dyne/cm2) for various durations using a cone-and-plate shear apparatus. We observed significant increases in the expression of key genes related to mitochondrial biogenesis and mitochondrial quality control as well as mtDNA content and mitochondrial mass under the shear stress conditions. Mitochondrial respiratory function was enhanced when cells were intermittently exposed to laminar shear stress for 72 hrs. Also, shear-exposed cells showed diminished glycolysis and decreased mitochondrial membrane potential (ΔΨm). Likewise, in in vivo experiments, mice that were subjected to a voluntary wheel running exercise for 5 weeks showed significantly higher mitochondrial content determined by en face staining in the conduit (greater and lesser curvature of the aortic arch and thoracic aorta) and muscle feed (femoral artery) arteries compared to the sedentary control mice. Interestingly, however, the mitochondrial biogenesis was not observed in the mesenteric artery. This region-specific adaptation is likely due to the differential blood flow redistribution during exercise in the different vessel beds.
Taken together, our findings suggest that exercise enhances mitochondrial biogenesis in vascular endothelium through a shear stress-dependent mechanism. Our findings may suggest a novel mitochondrial pathway by which a chronic exercise may be beneficial for vascular function.
β-catenin/TCF signaling regulates a varied set of cellular functions including development and remodeling. Fibronectin is a TCF-regulated gene that is highly expressed in arterial endothelium during atherosclerosis development and contributes to the pathophysiology of the disease. However, the activation of endothelial β-catenin/TCF signaling and its role in fibronectin expression in atherosclerosis are not currently known.
To assess the activity of β-catenin/TCF signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium.
Methods and Results
Histological staining identified preferential nuclear localization of β-catenin in the endothelium of atheroprone aorta prior to and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with β-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of β-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required β-catenin, TCF and the transcriptional co-activator CBP. Finally, we identified PECAM-1as a critical regulator of constitutive β-catenin and GSK-3β activities.
This data uncovers novel constitutive activation of the endothelial β-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.
β-catenin; TCF/LEF; endothelium; atherosclerosis; hemodynamics; fibronectin
Human immunodeficiency virus type 1 Tat exerts prominent angiogenic effects which may lead to a variety of vasculopathic conditions in AIDS patients. Because endothelial cells undergo prominent cytoskeletal rearrangement during angiogenesis, we investigated the specific effects of Tat on the endothelial cell actin cytoskeleton. Glutathione S-transferase (GST)-Tat, at a level of 200 ng/ml (equivalent to 52 ng of Tat/ml), caused stress fiber disassembly, peripheral retraction, and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells. At 600 ng of GST-Tat/ml (157 ng of Tat/ml), actin structures were lost, and severe cytoskeletal collapse occurred. In contrast, GST-Tat harboring mutations within either the cysteine-rich or basic domains exerted minimal effects on the endothelial cytoskeleton. HUVEC expressing a DsRed-Tat fusion protein displayed similar actin rearrangements, followed by actin collapse, whereas neighboring nontransfected cells retained normal actin structures. Because active mutants of p21-activated kinase 1 (PAK1) induce identical changes in actin dynamics, we hypothesized that Tat exerts its cytoskeletal effects through PAK1. GST-Tat activated PAK1 within 5 min, and adenovirus delivery of a kinase-dead PAK1 [PAK1(K298A)] completely prevented cytoskeletal collapse induced by GST-Tat or DsRed-Tat and also blocked downstream activation of c-Jun N-terminal kinase. Further, GST-Tat increased phosphorylation of the NADPH oxidase subunit p47phox and caused its rapid redistribution to membrane ruffles. PAK1(K298A) blocked p47phox phosphorylation, and interference with NADPH oxidase function through superoxide scavenging or through expression of a transdominant inhibitor, p67(V204A), prevented GST-Tat-induced alterations in the actin cytoskeleton. We conclude that Tat induces actin cytoskeletal rearrangements through PAK1 and downstream activation of the endothelial NADPH oxidase.
The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1−/−) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1−/− and wild type mice. Pak1−/− mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1−/− mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.
AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established.
The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress and endothelial function.
Methods and Results
AMPK activity, the markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65) were examined in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from AMPKα2 deficient mice. Compared to the wild type, acetylcholine (Ach)-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKα2−/− mice. Further, pretreatment of aorta with either superoxide dismutase or Tempol or apocynin significantly improved Ach-induced endothelium-dependent relaxation in AMPKα2−/− mice. Analysis of aortic endothelial cells from AMPKα2−/− mice and human umbilical vein endothelial cells (HUVECs) expressing dominant negative AMPK or AMPK α2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65), whereas AMPK activation by AICAR or over-expression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKα2 in LDL receptor knockout (LDLr−/−) strain markedly increased 26S proteasome activity, IκB degradation, NF-κB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, endothelial dysfunction, and atherosclerosis, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor..
We conclude that AMPKα2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way AMPK maintains the non-atherogenic and non-inflammatory phenotype of endothelial cells.
AMPK; NAD(P)H Oxidase; NF-κB; Proteasome
The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.