Mechanical loading and injury induce human myotubes to release neutrophil chemoattractants

The purpose of this study was to 1) test the hypothesis that skeletal muscle cells (myotubes) after mechanical loading and/or injury are a source of soluble factors that promote neutrophil chemotaxis and superoxide anion (O₂^–·) production and 2) determine whether mechanical loading and/or injury causes myotubes to release cytokines that are known to influence neutrophil responses [tumor necrosis factor- (TNF-), IL-8, and transforming growth factor-1 (TGF-1)]. Human myotubes were grown in culture and exposed to either a cyclic strain (0, 5, 10, 20, or 30% strain) or a scrape injury protocol. Protocols of 5, 10, and 20% strain did not cause injury, whereas 30% strain and scrape injury caused a modest and a high degree of injury, respectively. Conditioned media from strained myotubes promoted chemotaxis of human blood neutrophils and primed them for O₂^–· production in a manner that was dependent on a threshold of strain and independent from injury. Neutrophil chemotaxis, but not priming, progressively increased with higher magnitudes of strain. Conditioned media only from scrape-injured myotubes increased O₂^–· production from neutrophils. Concentrations of IL-8 and total TGF-1 in conditioned media were reduced by mechanical loading, whereas TNF- and active TGF-1 concentrations were unaffected. In conclusion, skeletal muscle cells after mechanical loading and injury are an important source of soluble factors that differentially influence neutrophil chemotaxis and the stages of neutrophil-derived reactive oxygen species production. Neutrophil responses elicited by mechanical loading, however, did not parallel changes in the release of IL-8, TGF-1, or TNF- from skeletal muscle cells. inflammation; cytokines; exercise; free radicals     

Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro

Endothelial cells actively participate in inflammatory events by regulating leukocyte recruitment via the expression of inflammatory genes such as E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and cyclooxygenase (COX)-2. In this study we showed by real-time RT-PCR that activation of human umbilical vein endothelial cells (HUVEC) by TNF- and IL-1 differentially affected the expression of these inflammatory genes. Combined treatment with TNF- and IL-1 resulted in nonadditive, additive, and even synergistic induction of expression of VCAM-1, IL-8, and IL-6, respectively. Overexpression of dominant-negative inhibitor B protein blocking NF-B signaling confirmed a major role of this pathway in controlling both TNF-- and IL-1-induced expression of most of the genes studied. Although dexamethasone exerted limited effects at 1 µM, the thioredoxin inhibitor MOL-294, which regulates the redox state of NF-B, mainly inhibited adhesion molecule expression. Its most pronounced effect was seen on VCAM-1 mRNA levels, especially in IL-1-activated endothelium. One micromolar RWJ-67657, an inhibitor of p38 MAPK activity, diminished TNF-- and IL-1-induced expression of IL-6, IL-8, and E-selectin but had little effect on VCAM-1 and ICAM-1. Combined treatment of HUVEC with MOL-294 and RWJ-67657 resulted in significant blocking of the expression of E-selectin, IL-6, IL-8, and COX-2. The inhibitory effects were much stronger than those observed with single drug treatment. Application of combinations of drugs that affect multiple targets in activated endothelial cells may therefore be considered as a potential new therapeutic strategy to inhibit inflammatory disease activity. inflammatory gene expression; anti-inflammatory drugs; pharmacology; combination treatment     

TNF-alpha is a mitogen in skeletal muscle

Emerging evidence suggests that tumor necrosis factor (TNF)- plays a role in muscle repair. To determine whether TNF- modulates satellite cell proliferation, the current study evaluated TNF- effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF- increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2'-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF- stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF- stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c-fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF- activates satellite cells to enter the cell cycle and accelerates G₁-to-S phase transition, and these actions may involve activation of early response genes via SRF. cytokine; cell cycle; satellite cells; serum response factor; c-fos     

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF- plus cycloheximide (TNF-/CHX). Specific inhibition of NF-B by the recombinant adenoviral vector containing the IB superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-B activity and increased the sensitivity to TNF-/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-B activity and resulted in increased resistance to TNF-/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-/CHX-induced apoptosis, at least partially, through the activation of NF-B signaling in IECs. polyamines; -difluoromethylornithine; X-linked inhibitor of apoptosis protein; IB     

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta

Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell     

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway

Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c     

Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins

Heterotrimeric G_i proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G_i proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of G_i2 or G_i1/3 protein in G_i2-knockout (G_i2–/–) or G_i1/3-knockout (G_i1/3–/–) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (M) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF- and IFN- in splenocytes from G_i2–/– mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from G_i1/3–/– mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B₂ (TxB₂) production was decreased in M harvested from G_i2–/– mice. Also, LPS-induced production of IL-10 and TxB₂ was decreased in M from G_i1/3–/– mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in G_i2–/– mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated G_i2–/– mice compared with WT mice. These data suggest that G_i proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. G_i protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors     

Estrogen receptor-alpha predominantly mediates the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage

Although 17-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)- or ER- and which signaling pathways are involved in such 17-estradiol effects. Utilizing ER-- or ER--specific agonists, this study examined the role of ER- and ER- in 17-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), the ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF- production and activation of MAPK and NF-B were measured. Macrophage IL-6 and TNF- production and MAPK activation were decreased, whereas NF-B activity was increased, following trauma-hemorrhage. PPT or 17-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER- plays the predominant role in mediating the salutary effects of 17-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-B signaling pathways. shock; mitogen-activated protein kinase; nuclear factor-B; propyl pyrazole triol; diarylpropionitrile     

Dominant-negative PKC-epsilon impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini

We investigated the involvement of PKC- in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC- cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 µM, 2–15 min) significantly (P 0.05) increased PKC- recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC- association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC- in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and -hexosaminidase. The chemical inhibitor GF-109203X (10 µM, 3 h), which inhibits PKC-, -, -, and -, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 µM, 3 h), which inhibits only PKC- and -. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC- significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC- transduction suppressed its carbachol-stimulated release. We propose that DN-PKC- alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis. lacrimal gland; acinar epithelial cell; exocytosis; polymeric immunoglobulin A receptor     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (, I, II, and ), novel [, , , and µ (also known as PKD),], and atypical ( and /), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms , I, and and complete translocation of PKC and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKC and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms , I, and were dispensable with regard to these same phenomena. signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium     

PKC zeta participates in activation of inflammatory response induced by enteropathogenic E. coli

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-B activation but that other signaling molecules such as PKC may be involved. The aim of this study was to determine whether PKC is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC pseudosubstrate (MYR-PKC-PS), or transient expression of a nonfunctional PKC significantly suppressed EPEC-induced IB phosphorylation. Although PKC can activate ERK, MYR-PKC-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC can regulate NF-B activation by interacting with and activating IB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC-IKK complexes from infected intestinal epithelial cells and recombinant IB as a substrate showed a 2.5-fold increase in IB phosphorylation. PKC can also regulate NF-B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC-PS, suggesting that other signaling events may be involved in this particular arm of NF-B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC and ERK that lead to NF-B activation, thus ensuring the proinflammatory response. inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C; IB kinase; extracellular signal-regulated kinase     

An osteoclastic protein-tyrosine phosphatase may play a role in differentiation and activity of human monocytic U-937 cell-derived, osteoclast-like cells

This study investigated if an osteoclastic protein-tyrosine phosphatase (PTP), PTP-oc, plays a role in the functional activity and differentiation of osteoclastic cells by determining the effects of overexpression of wild-type (WT)- or phosphatase-deficient (PD)-PTP-oc on bone resorption activity and differentiation of human promyelomonocytic U-937 cells, which could be induced to differentiate into "osteoclast-like" cells by phorbol ester/1,25(OH)₂D₃ treatment. U-937 cells overexpressing WT- or PD-PTP-oc were produced with a transposon-based vector. The size and depth of resorption pits created by WT-PTP-oc-overexpressing osteoclast-like cells were greater, while those by PD-PTP-oc-overexpressing osteoclast-like cells were less, than those created by control osteoclast-like cells. Overexpression of WT-PTP-oc also enhanced, while overexpression of PD-PTP-oc suppressed, their differentiation into osteoclast-like cells. Overexpression of WT-PTP-oc increased apoptosis and proliferation of U-937 cells, and overexpression of PD-PTP-oc reduced cell proliferation. Cells overexpressing WT-PTP-oc has also led to greater c-Src and NF- activation, whereas cells overexpressing PD-PTP-oc resulted in less c-Src and NF- activation. c-Src activation and NF- activation each correlated with resorption activity and differentiation into osteoclast-like cells. In summary, these results show that 1) PTP-oc regulates both the activity and the differentiation of osteoclast-like cells derived from U-937 cells; 2) PTP-oc enzymatic activity is important to these processes; 3) high PTP-oc enzymatic activity caused an increase in U-937 cell apoptosis and proliferation, leading to no significant changes in the number of viable cells; and 4) some of the PTP-oc actions are mediated in part by the c-Src and/or NF- pathways. osteoclast; resorption; nuclear factor-; c-Src     

Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with ₁-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of ₁-integrin signaling using laminin inhibited the increased association of MMP-2 with ₁-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with ₁ integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase     

Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-kappaB in endothelial cells

NF-B signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-B activity is transient, our earlier studies have demonstrated a persistent activation of NF-B in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-B signaling kinases (IKK and ) that may be responsible for the sustained activation of NF-B in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKK, but it is also persistently induced for a prolonged time frame. The IKK activity (both and ) is blocked by apocynin (400 µM), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 µM), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-B activation selectively in low shear-induced endothelial cells. upstream B kinases; laminar shear stress; oxidative stress; atherogenesis; reactive oxygen species     

HO-2 provides endogenous protection against oxidative stress and apoptosis caused by TNF-alpha in cerebral vascular endothelial cells

Tumor necrosis factor- (TNF-) causes oxidative stress and apoptosis in a variety of cell types. Heme oxygenase (HO) degrades heme to bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator, and a vasodilator. Newborn pig cerebral microvascular endothelial cells (CMVEC) highly express constitutive HO-2. We investigated the role of HO-2 in protection against TNF--induced apoptosis in cerebral vascular endothelium. In CMVEC from mice and newborn pigs, 15 ng/ml TNF- alone, or with 10 µg/ml cycloheximide (CHX) caused apoptosis detected by nuclear translocation of p65 NF-B, caspase-3 activation, DNA fragmentation, cell-cell contact destabilization, and cell detachment. TNF- did not induce HO-1 expression in CMVEC. CMVEC from HO-2 knockout mice showed greater sensitivity to apoptosis caused by serum deprivation and TNF- than did wild-type mice. TNF- increased reactive oxygen species generation, including hydrogen peroxide and superoxide radicals, as detected by dihydrorhodamine-123 and dihydroethidium. The TNF- response was inhibited by superoxide dismutase and catalase suggesting apoptosis is oxidative stress related. Inhibition of endogenous HO-2 in newborn pig CMVEC increased oxidative stress and exaggerated apoptosis caused by serum deprivation and TNF-. In HO-1-overexpressing CMVEC (HO-1 selective induction by cobalt portophyrin), TNF- did not cause apoptosis. A CO-releasing compound, CORM-A1, and bilirubin blocked TNF--induced reactive oxygen species accumulation and apoptosis consistent with the antioxidant and antiapoptotic roles of the end products of HO activity. We conclude that HO-2 is critical for protection of cerebrovascular endothelium against apoptotic changes induced by oxidative stress and cytokine-mediated inflammation. carbon monoxide; bilirubin; vascular injury; reactive oxygen species; heme oxygenase; cycloheximide     

Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-alpha

Angiotensin II (ANG II) has been etiologically linked to vascular disease; however, its role in the alterations of endothelial function that occur in vascular disorders is not completely understood. Matrix metalloproteinases (MMPs) and proinflammatory cytokines are involved in the pathological remodeling of blood vessels that occurs in vascular disease. In this study we evaluated the effects of ANG II on tumor necrosis factor (TNF)- and MMP-2 production in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with ANG II (0.1–10 µM) for 24 h, in the presence or absence of antagonists of ANG II type 1 (AT₁R) and type 2 (AT₂R) receptors, and the production and release of TNF- and MMP-2 were assessed. ANG II increased TNF- mRNA and protein expression and the release of bioactive TNF-. Moreover, ANG II induced MMP-2 release and reduced the secretion of tissue inhibitor of MMP (TIMP)-2 from endothelial cells. To elucidate whether endogenous TNF- could mediate the effects of ANG II on MMP-2 release, cells were pretreated with anti-TNF- neutralizing antibodies or pentoxifylline (an inhibitor of TNF- synthesis). TNF- inhibition prevented the secretion of MMP-2 induced by ANG II. Furthermore, AT₁R antagonism with candesartan prevented the formation of MMP-2 and TNF- and the reduction of TIMP-2 induced by ANG II. These results indicate that ANG II, via AT₁R, modulates the secretion of TNF- and MMP-2 from endothelial cells and that TNF- mediates the effects of ANG II on MMP-2 release. remodeling; vasoactive mediators; inflammation     

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and ₁-subunits (BK-₁), or both - and ₄-subunits (BK-₄) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-₁ within a similar concentration range. Because methAEA could potentiate BK-, BK-₁, and BK-₄ with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-₁ did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB₁ antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB₂ agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener     

TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF- mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-, when added to these cells in the presence of serum-containing media, increases p73 expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH₂-terminally deleted p73 plasmid results in markedly decreased TNF--induced apoptosis. Thus p73 is likely a mediator of the apoptotic effect of TNF- in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies. atherosclerosis; inflammation; plaque