Prevention of aortic fibrosis by N-acetyl-seryl-aspartyl-lysyl-proline in angiotensin II-induced hypertension

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-beta1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-beta1 and inhibition of Smad2 phosphorylation.     

Characterization and localization of Ac-SDKP receptor binding sites using 125I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand, (125)I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. (125)I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 +/- 198 fmol/mg protein; dissociation constant: 3.3 +/- 0.6 nM). (125)I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 +/- 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 +/- 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 microM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 +/- 0.4, ET-1: 19.9 +/- 1.2, ET-1+SDKP: 7.7 +/- 0.4, ET-1+Hpp-Aca-SDKP: 9.7 +/- 0.1 microg/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific (125)I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Regulation of alternative macrophage activation by galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.     

Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-kappaB, p38, and reactive oxygen species

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.     

ANG II stimulates phospholipase D through PKCzeta activation in VSMC: implications in adhesion, spreading, and hypertrophy

ANG II stimulates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C-zeta (PKCzeta) plays a central role in the regulation of cell survival and proliferation. This study was conducted to determine the relationship between ANG II-induced activation of PKCzeta and PLD and their implication in VSMC adhesion, spreading, and hypertrophy. ANG II stimulated PKCzeta activity with maximal activation at 30 s followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKCzeta activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKCzeta decreased ANG II-induced PLD activity. Moreover, depletion of PKCzeta with selective antisense oligonucleotides also decreased ANG II-induced PLD activity. Interaction between PLD2 and PKCzeta in VSMC was detected by coimmunoprecipitation. ANG II-induced PLD activity was inhibited by the primary alcohol n-butanol but not the tertiary alcohol t-butanol. The functional significance of PKCzeta and PLD2 in VSMC adhesion, spreading, and hypertrophy was investigated. Inhibition of PKCzeta and PLD2 activity or expression attenuated VSMC adhesion to collagen I and ANG II-induced cell spreading and hypertrophy. These results demonstrate that ANG II-induced PLD activation is regulated by PKCzeta and suggest a crucial role of PKCzeta-dependent PLD2 in VSMC functions such as adhesion, spreading, and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension.     

ANG II-induced cardiac molecular and cellular events: role of aldosterone

Chronic elevation of circulating ANG II is associated with cardiac remodeling in patients with hypertension and heart failure. The underlying mechanisms, however, are not completely defined. Herein, we studied ANG II-induced molecular and cellular events in the rat heart as well as their links to the redox state. We also addressed the potential contribution of aldosterone (ALDO) on ANG II-induced cardiac remodeling. In ANG II-treated rats, and compared with controls, we found: 1) the expression of proinflammatory/profibrogenic mediators was significantly increased in the perivascular space and at the sites of microscopic injury in both ventricles; 2) macrophages and myofibroblasts were primary repairing cells at these sites, together with increased fibrillar collagen volume; 3) apoptotic macrophages and myofibroblasts were evident at the same sites; 4) NADPH oxidase (gp91phox) was significantly enhanced at these regions and primarily expressed by macrophages, whereas superoxide dismutase and catalase levels remained unchanged; 5) plasma 8-isoprostane levels were significantly increased; and 6) blood pressure was significantly elevated. Losartan treatment completely prevented cardiac oxidative stress as well as molecular/cellular responses and normalized blood pressure. Spironolactone treatment partially suppressed the cardiac inflammatory/fibrogenic responses and redox state. Thus chronic elevation of circulating ANG II is accompanied by a proinflammatory/profibrogenic phenotype involving vascular and myocardial remodeling in both ventricles. Enhanced reactive oxygen species production at these sites and increased plasma 8-isoprostane indicate the involvement of oxidative stress in ANG II-induced cardiac injury. ALDO contributes, in part, to ANG II-induced cardiac molecular and cellular responses.     

Inhibition of LPS-induced cytokines by Bcl-xL in a murine macrophage cell line

The antiapoptotic molecule Bcl-xL has been implicated in the differentiation and survival of activated macrophages in inflammatory conditions. In this report, the role of Bcl-xL in LPS-induced cytokine gene expression and secretion was studied. Bcl-xL-transfected RAW 264 macrophages were protected from gliotoxin-induced apoptosis, indicating the presence of functional Bcl-xL. Overexpression of Bcl-xL in this macrophage cell line was also associated with a marked inhibition of LPS-induced TNF-alpha, JE/monocyte chemoattractant protein 1, and macrophage inflammatory protein 2 secretion. Inhibition of LPS-induced cytokine secretion was paralleled by a decrease in levels of steady-state mRNA for the above cytokines and for IL-1beta. Decreased production of TNF-alpha in Bcl-xL transfectants was not due to increased mRNA degradation, as the mRNA half-lives were the same in Bcl-xL transfectants and control macrophages. Although the composition of NF-kappaB complexes detected by EMSA and supershift analysis in nuclear lysates derived from Bcl-xL transfectants and control cells was indistinguishable, LPS-induced inhibitory kappaBalpha degradation, as well as NF-kappaB binding and AP-1 activation, were slightly decreased by ectopic expression of Bcl-xL. More strikingly, LPS-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase was strongly repressed by Bcl-xL overexpression, offering a possible mechanism for the inhibition of LPS-induced cytokine production. These data provide the first evidence for a novel role for Bcl-xL as an anti-inflammatory mediator in macrophages.     

Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase

Reduced insulin sensitivity is a key factor in the pathogenesis of type 2 diabetes and hypertension. Skeletal muscle insulin resistance is particularly important for its major role in insulin-mediated glucose disposal. Angiotensin II (ANG II) is integral in regulating blood pressure and plays a role in the pathogenesis of hypertension. In addition, we have documented that ANG II-induced skeletal muscle insulin resistance is associated with generation of reactive oxygen species (ROS). However, the linkage between ROS and insulin resistance in skeletal muscle remains unclear. To explore potential mechanisms, we employed the transgenic TG(mRen2)27 (Ren-2) hypertensive rat, which harbors the mouse renin transgene and exhibits elevated tissue ANG II levels, and skeletal muscle cell culture. Compared with Sprague-Dawley normotensive control rats, Ren-2 skeletal muscle exhibited significantly increased oxidative stress, NF-kappaB activation, and TNF-alpha expression, which were attenuated by in vivo treatment with an angiotensin type 1 receptor blocker (valsartan) or SOD/catalase mimetic (tempol). Moreover, ANG II treatment of L6 myotubes induced NF-kappaB activation and TNF-alpha production and decreased insulin-stimulated Akt activation and GLUT-4 glucose transporter translocation to plasma membranes. These effects were markedly diminished by treatment of myotubes with valsartan, the antioxidant N-acetylcysteine, NADPH oxidase-inhibiting peptide (gp91 ds-tat), or NF-kappaB inhibitor (MG-132). Similarly, NF-kappaB p65 small interfering RNA reduced NF-kappaB p65 subunit expression and nuclear translocation and TNF-alpha production but improved insulin-stimulated phosphorylation (Ser(473)) of Akt and translocation of GLUT-4. These findings suggest that NF-kappaB plays an important role in ANG II/ROS-induced skeletal muscle insulin resistance.     

ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression

In addition to well-documented vascular growth-promoting effects, ANG II exerts proapoptotic effects that are poorly understood. IGF-1 is a potent survival factor for human vascular smooth muscle cells (hVSMC), and its antiapoptotic effects are mediated via the IGF-1 receptor (IGF-1R) through a signaling pathway involving phosphatidylinositol 3-kinase and Akt. We hypothesized that there would be cross talk between ANG II proapoptotic effects and IGF-1 survival effects in hVSMC. To investigate ANG II-induced apoptosis and the potential involvement of IGF-1, we exposed quiescent and nonquiescent hVSMC to ANG II. ANG II induced apoptosis only in nonquiescent cells but stimulated hypertrophy in quiescent cells. ANG II-induced apoptosis was characterized by marked inhibition of Akt phosphorylation and stimulation of membrane Fas ligand (FasL) expression, caspase-8 activation, and a reduction in soluble FasL expression. Adenovirally mediated overexpression of Akt rescued hVSMC from ANG II-induced apoptosis. IGF-1R activation increased Akt phosphorylation and soluble FasL expression, and these effects were completely blocked by coincubating hVSMC with ANG II. In conclusion, ANG II-induced apoptosis of hVSMC is characterized by marked inhibition of Akt phosphorylation and stimulation of an extrinsic cell death signaling pathway via upregulation of membrane FasL expression, caspase-8 activation, and a reduction in soluble FasL expression. Furthermore, ANG II antagonizes the antiapoptotic effect of IGF-1 by blocking its ability to increase Akt phosphorylation and soluble FasL. These findings provide novel insights into ANG II-induced apoptotic signaling and have significant implication for understanding ANG II-induced remodeling in hypertension and atherosclerosis.     

IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production.     

Hexarelin suppresses cardiac fibroblast proliferation and collagen synthesis in rat

Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Hexarelin is a synthetic growth hormone secretagogue (GHS), which possesses a variety of cardiovascular protective activities mediated via the GHS receptor (GHSR), including improving cardiac dysfunction and remodeling. The cellular and molecular mechanisms underlying the effect of GHS on cardiac fibrosis are, however, not clear. In this report, cultured cardiac fibroblasts from 8-day-old rats were stimulated with ANG II or FCS to induce proliferation. The fibroblast proliferation and DNA and collagen synthesis were evaluated utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, (3)H-thymidine incorporation, and (3)H-proline incorporation. The level of mRNA of transforming growth factor (TGF)-beta was evaluated by RT-PCR, and the active TGF-beta1 release from cardiac fibroblasts was evaluated by ELISA. The level of cellular cAMP was measured by radioimmunoassay. In addition, the effects of 3,7-dimethyl-l-propargylxanthine (DMPX; a specific adenosine receptor A(2)R antagonist) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a specific A(1)R antagonist) were tested. It was found that incubation with 10(-7) mol/l hexarelin for 24 h 1) inhibited the ANG II-induced proliferation and collagen synthesis and the 5% FCS- and TGF-beta-induced increase of DNA synthesis in cardiac fibroblast and 2) reduced ANG II-induced upregulation of TGF-beta mRNA expression and active TGF-beta1 release from fibroblasts. Hexarelin increased the cellular level of cAMP in cardiac fibroblasts. DMPX (10(-8) mol/l) but not DPCPX abolished the effect of hexarelin on cardiac fibroblast DNA synthesis. It is concluded that hexarelin inhibits DNA and collagen synthesis and proliferation of cardiac fibroblasts through activation of both GHSR and A(2)R and diminishment of ANG II-induced increase in TGF-beta expression and release.     

TGF-beta 1 and IFN-gamma direct macrophage activation by TNF-alpha to osteoclastic or cytocidal phenotype

TNF-related activation-induced cytokine (TRANCE; also called receptor activator of NF-kappaB ligand (RANKL), osteoclast differentiation factor (ODF), osteoprotegerin ligand (OPGL), and TNFSF11) induces the differentiation of progenitors of the mononuclear phagocyte lineage into osteoclasts in the presence of M-CSF. Surprisingly, in view of its potent ability to induce inflammation and activate macrophage cytocidal function, TNF-alpha has also been found to induce osteoclast-like cells in vitro under similar conditions. This raises questions concerning both the nature of osteoclasts and the mechanism of lineage choice in mononuclear phagocytes. We found that, as with TRANCE, the macrophage deactivator TGF-beta(1) strongly promoted TNF-alpha-induced osteoclast-like cell formation from immature bone marrow macrophages. This was abolished by IFN-gamma. However, TRANCE did not share the ability of TNF-alpha to activate NO production or heighten respiratory burst potential by macrophages, or induce inflammation on s.c. injection into mice. This suggests that TGF-beta(1) promotes osteoclast formation not only by inhibiting cytocidal behavior, but also by actively directing TNF-alpha activation of precursors toward osteoclasts. The osteoclast appears to be an equivalent, alternative destiny for precursors to that of cytocidal macrophage, and may represent an activated variant of scavenger macrophage.     

Requirements for apoptotic cell contact in regulation of macrophage responses

An important consequence of macrophage engulfment of apoptotic cells is suppression of inflammatory responses, which was first defined by assay of TNF-alpha release stimulated by LPS. These effects are apparently mediated in part by paracrine effects of TGF-beta released by the subset of stimulated macrophages that ingest apoptotic cells, which suppresses neighboring cells. However, the apoptotic cell-derived signal that stimulates TGF-beta release, and the nature of any additional signals required for the anti-inflammatory response remain poorly defined. In this study, we investigate the requirements for apoptotic cell engagement of macrophage surface receptors in these responses. We show that the apoptotic cell receptors CD36 and alphavbeta3 contribute to apoptotic cell phagocytosis by mouse macrophages, but are not essential for anti-inflammatory responses, suggesting that the mechanisms of response and phagocytosis are separate. In further defining requirements for response, we confirm the importance of TGF-beta in suppression by apoptotic cells, and identify an additional level of control of these effects. We show that LPS-stimulated mouse macrophage TNF-alpha release is only suppressed if macrophages have first contacted apoptotic cells, and hence, bystander macrophages are refractory to TGF-beta released by phagocytosing macrophages. We conclude that the profound suppression of LPS-driven TNF-alpha release by macrophage populations requires hitherto obscure contact-dependent licensing of macrophage responsiveness to TGF-beta by apoptotic cells.     

Constitutive activation of phosphatidylinositol 3-kinase signaling pathway down-regulates TLR4-mediated tumor necrosis factor-alpha release in alveolar macrophages from asymptomatic HIV-positive persons in vitro

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinase (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 microg/ml) resulted in lower TNF-alpha release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-alpha release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3beta (GSK-3beta) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3beta phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3beta (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-alpha release in HIV+ human macrophages and impair host cell response to infectious challenge.     

Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident,inflammatory, and activated macrophages

Recent evidence has implicated galectins and their carbohydrate ligands as master regulators of the inflammatory response. Galectin-1, a member of this family, has shown specific anti-inflammatory and immunoregulatory effects. To gain insight into the potential mechanisms involved in these effects, we investigated the effects of galectin-1 in L-arginine metabolism of peritoneal rat macrophages. Pretreatment of macrophages with galectin-1 resulted in a dose- and time-dependent inhibition of lipopolysaccharide-induced nitric oxide (NO) production, accompanied by a decrease in inducible nitric oxide synthase (iNOS) expression (the classic pathway of L-arginine). On the other hand, galectin-1 favored the balance toward activation of L-arginase, the alternative metabolic pathway of L-arginine. Inhibition of NO production was not the result of increased macrophage apoptosis because addition of this beta-galactoside-binding protein to macrophages under the same experimental conditions did not affect the apoptotic threshold of these cells. To understand how endogenous galectin-1 is regulated in macrophages under inflammatory stress, we finally explored the ultrastructural distribution, expression, and secretion of galectin-1 in resident, inflammatory, and activated macrophages. This study provides an alternative cellular mechanism based on the modulation of L-arginine metabolism to understand the molecular basis of the anti-inflammatory properties displayed by this carbohydrate-binding protein.     

Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation

Early inflammatory events include cytokine release, activation, and rapid accumulation of neutrophils, with subsequent recruitment of mononuclear cells. The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway plays a central role in regulating a wide range of inflammatory responses in many different cells. A murine model of mild LPS-induced lung inflammation was developed to investigate the role of the p38 MAPK pathway in the initiation of pulmonary inflammation. A novel p38 MAPK inhibitor, M39, was used to determine the functional consequences of p38 MAPK activation. In vitro exposure to M39 inhibited p38 MAPK activity in LPS-stimulated murine and human neutrophils and macrophages, blocked TNF-alpha and macrophage inflammatory protein-2 (MIP-2) release, and eliminated migration of murine neutrophils toward the chemokines MIP-2 and KC. In contrast, alveolar macrophages required a 1000-fold greater concentration of M39 to block release of TNF-alpha and MIP-2. Systemic inhibition of p38 MAPK resulted in significant decreases in the release of TNF-alpha and neutrophil accumulation in the airspaces following intratracheal administration of LPS. Recovery of MIP-2 and KC from the airspaces was not affected by inhibition of p38 MAPK, and accumulation of mononuclear cells was not significantly reduced. When KC was instilled as a proinflammatory stimulus, neutrophil accumulation was significantly decreased by p38 MAPK inhibition independent of TNF-alpha or LPS. Together, these results demonstrate a much greater dependence on the p38 MAPK cascade in the neutrophil when compared with other leukocytes, and suggest a means of selectively studying and potentially modulating early inflammation in the lung.     

Involvement of cytochrome P-450 1B1 in renal dysfunction, injury, and inflammation associated with angiotensin II-induced hypertension in rats

We investigated the contribution of cytochrome P-450 1B1 (CYP1B1) to renal dysfunction and organ damage associated with ANG II-induced hypertension in rats. ANG II (300 ng·kg(-1)·min(-1)) or vehicle were infused for 2 wk, with daily injections of a selective CYP1B1 inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS; 300 μg/kg ip), or its vehicle. ANG II increased blood pressure and renal CYP1B1 activity that were prevented by TMS. ANG II also increased water intake and urine output, decreased glomerular filtration rate, increased urinary Na(+) and K(+) excretion, and caused proteinuria, all of which were prevented by TMS. ANG II infusion caused hypertrophy, endothelial dysfunction, and increased reactivity of renal and interlobar arteries to vasoconstrictor agents and renal vascular resistance and interstitial fibrosis as indicated by accumulation of α-smooth muscle actin, fibronectin, and collagen, and inflammation as indicated by increased infiltration of CD-3(+) cells; these effects were inhibited by TMS. ANG II infusion also increased production of reactive oxygen species (ROS) and activities of NADPH oxidase, ERK1/2, p38 MAPK, and c-Src that were prevented by TMS. TMS alone had no effect on any of the above parameters. These data suggest that CYP1B1 contributes to the renal pathophysiological changes associated with ANG II-induced hypertension, most likely via increased ROS production and activation of ERK1/2, p38 MAPK, and c-Src and that CYP1B1 could serve as a novel target for treating renal disease associated with hypertension.     

Central estrogen inhibition of angiotensin II-induced hypertension in male mice and the role of reactive oxygen species

It has been shown that reactive oxygen species (ROS) contribute to the central effect of ANG II on blood pressure (BP). Recent studies have implicated an antihypertensive action of estrogen in ANG II-infused female mice. The present study used in vivo telemetry recording and in vitro living mouse brain slices to test the hypothesis that the central activation of estrogen receptors in male mice inhibits ANG II-induced hypertension via the modulation of the central ROS production. In male wild-type mice, the systemic infusion of ANG II induced a significant increase in BP (Delta30.1 +/- 2.5 mmHg). Either central infusion of Tempol or 17beta-estradiol (E2) attenuated the pressor effect of ANG II (Delta10.9 +/- 2.3 and Delta4.5 +/- 1.4 mmHg), and the protective effect of E2 was prevented by the coadministration of an estrogen receptor, antagonist ICI-182780 (Delta23.6 +/- 3.1 mmHg). Moreover, the ganglionic blockade on day 7 after the start of ANG II infusions resulted in a smaller reduction of BP in central Tempol- and in central E2-treated males, suggesting that estrogen inhibits the central ANG II-induced increases in sympathetic outflow. In subfornical organ slices, the application of ANG II resulted in a 21.5 +/- 2.5% increase in ROS production. The coadministration of irbesartan, an ANG II type 1 receptor antagonist, or the preincubation of brain slices with Tempol blocked ANG II-induced increases in ROS production (-1.8 +/- 1.6% and -1.0 +/- 1.8%). The ROS response to ANG II was also blocked by E2 (-3.2 +/- 2.4%). The results suggest that the central actions of E2 are involved in the protection from ANG II-induced hypertension and that estrogen modulation of the ANG II-induced effects may involve interactions with ROS production.