Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury.

[Figure: see text].

Nrg1ß Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy.

OBJECTIVE: Remote ischemic preconditioning (RIPC) is an intervention process where the application of multiple cycles of short ischemia/reperfusion (I/R) in a remote vascular bed provides protection against I/R injury. However, the identity of the specific RIPC factor and the mechanism by which RIPC alleviates I/R injury remains unclear. Here, we have investigated the identity and the mechanism by which the RIPC factor provides protection. APPROACH AND RESULTS: Using fluorescent in situ hybridization and immunofluorescence, we found that RIPC induces Nrg1ß expression in the endothelial cells, which is secreted into the serum. Whereas, RIPC protected against myocardial apoptosis and infarction, treatment with neutralizing-Nrg1 antibodies abolished the protective effect of RIPC. Further, increased superoxide anion generated in RIPC is required for Nrg1 expression. Improved myocardial perfusion and nitric oxide production were achieved by RIPC as determined by contrast echocardiography and electron spin resonance. However, treatment with neutralizing-Nrg1ß antibody abrogated these effects, suggesting Nrg1ß is a RIPC factor. ErbB2 (Erb-B2 receptor tyrosine kinase 2) is not expressed in the adult murine cardiomyocytes, but expressed in the endothelial cells of heart which is degraded in I/R. RIPC-induced Nrg1ß interacts with endothelial ErbB2 and thereby prevents its degradation. Mitochondrial Trx2 (thioredoxin) is degraded in I/R, but rescue of ErbB2 by Nrg1ß prevents Trx-2 degradation that decreased myocardial apoptosis in I/R. CONCLUSIONS: Nrg1ß is a RIPC factor that interacts with endothelial ErbB2 and prevents its degradation, which in turn prevents Trx2 degradation due to phosphorylation and inactivation of ATG5 (autophagy-related 5) by ErbB2. Nrg1ß also restored loss of eNOS (endothelial nitric oxide synthase) function in I/R via its interaction with Src.

Thioredoxin Decreases Anthracycline Cardiotoxicity, But Sensitizes Cancer Cell Apoptosis.

Cardiotoxicity is a major limitation for anthracycline chemotherapy although anthracyclines are potent antitumor agents. The precise mechanism underlying clinical heart failure due to anthracycline treatment is not fully understood, but is believed to be due, in part, to lipid peroxidation and the generation of free radicals by anthracycline-iron complexes. Thioredoxin (Trx) is a small redox-active antioxidant protein with potent disulfide reductase properties. Here, we present evidence that cancer cells overexpressing Trx undergo enhanced apoptosis in response to daunomycin. In contrast, cells overexpressing redox-inactive mutant Trx were not effectively killed. However, rat embryonic cardiomyocytes (H9c2 cells) overexpressing Trx were protected against daunomycin-mediated apoptosis, but H9c2 cells with decreased levels of active Trx showed enhanced apoptosis in response to daunomycin. We further demonstrate that increased level of Trx is specifically effective in anthracycline toxicity, but not with other topoisomerase II inhibitors such as etoposide. Collectively these data demonstrate that whereas high levels of Trx protect cardiomyocytes against anthracycline toxicity, it potentiates toxicity of anthracyclines in cancer cells.

Thioredoxin protects mitochondrial structure, function and biogenesis in myocardial ischemia-reperfusion via redox-dependent activation of AKT-CREB- PGC1α pathway in aged mice.

Aging is an independent risk factor for cardiovascular diseases, such as myocardial infarction due to ischemia-reperfusion injury (I/R) of the heart. Cytosolic thioredoxin (Trx) is a multifunctional redox protein which has antioxidant and protein disulfide reducing properties. We hypothesized that high levels of Trx will protect against multifactorial disease such as myocardial infarction due to I/R injury in aged mice. Aged mice overexpressing human Trx (Trx-Tg), mice expressing redox-inactive mutant of human Trx (dnTrx-Tg) and non-transgenic litter-mates (NT) were subjected to I/R (60/30 min), and cardiac function, mitochondrial structure and function, and biogenesis involving PGC1α pathway were evaluated in these mice. While aged Trx-Tg mice were protected from I/R-induced reduction in ejection fraction (EF) and fractional shortening (FS), had smaller infarct with decreased apoptosis and preserved mitochondrial function, aged dnTrx-Tg mice showed enhanced myocardial injury and mitochondrial dysfunction. Further, Trx-Tg mice were protected from I/R induced loss of PGC1α, ACO2, MFN1 and MFN2 in the myocardium. The dnTrx-Tg mice were highly sensitive to I/R induced apoptosis. Overall, our study demonstrated that the loss of Trx redox balance in I/R in aged NT or dnTrx-Tg mice resulted in decreased PGC1α expression that decreased mitochondrial gene expression with increased myocardial apoptosis. High levels of Trx, but not mitochondrial thioredoxin (Trx-2) maintained Trx redox balance in I/R resulting in increased PGC1α expression via AKT/CREB activation upregulating mitochondrial gene expression and protection against I/R injury.

Short-duration hyperoxia causes genotoxicity in mouse lungs: protection by volatile anesthetic isoflurane.

High concentrations of oxygen (hyperoxia) are routinely used during anesthesia, and supplemental oxygen is also administered in connection with several other clinical conditions. Although prolonged hyperoxia is known to cause acute lung injury (ALI), whether short-duration hyperoxia causes lung toxicity remains unknown. We exposed mice to room air (RA or 21% O2) or 60% oxygen alone or in combination with 2% isoflurane for 2 h and determined the expression of oxidative stress marker genes, DNA damage and DNA repair genes, and expression of cell cycle regulatory proteins using quantitative PCR and Western analyses. Furthermore, we determined cellular apoptosis using TUNEL assay and assessed the DNA damage product 8-hydroxy-2'-deoxyguanosine (8-Oxo-dG) in the urine of 60% hyperoxia-exposed mice. Our study demonstrates that short-duration hyperoxia causes mitochondrial and nuclear DNA damage and that isoflurane abrogates this DNA damage and decreases apoptosis when used in conjunction with hyperoxia. In contrast, isoflurane mixed with RA caused significant 8-Oxo-dG accumulations in the mitochondria and nucleus. We further show that whereas NADPH oxidase is a major source of superoxide anion generated by isoflurane in normoxia, isoflurane inhibits superoxide generation in hyperoxia. Additionally, isoflurane also protected the mouse lungs against ALI (95% O2 for 36-h exposure). Our study established that short-duration hyperoxia causes genotoxicity in the lungs, which is abrogated when hyperoxia is used in conjunction with isoflurane, but isoflurane alone causes genotoxicity in the lung when delivered with ambient air.

Role of Thioredoxin in Age-Related Hypertension.

PURPOSE OF REVIEW: Although the roles of oxidant stress and redox perturbations in hypertension have been the subject of several reviews, role of thioredoxin (Trx), a major cellular redox protein in age-related hypertension remains inadequately reviewed. The purpose of this review is to bring readers up-to-date with current understanding of the role of thioredoxin in age-related hypertension. RECENT FINDINGS: Age-related hypertension is a major underlying cause of several cardiovascular disorders, and therefore, intensive management of blood pressure is indicated in most patients with cardiovascular complications. Recent studies have shown that age-related hypertension was reversed and remained lowered for a prolonged period in mice with higher levels of human Trx (Trx-Tg). Additionally, injection of human recombinant Trx (rhTrx) decreased hypertension in aged wild-type mice that lasted for several days. Both Trx-Tg and aged wild-type mice injected with rhTrx were normotensive, showed increased NO production, decreased arterial stiffness, and increased vascular relaxation. These studies suggest that rhTrx could potentially be a therapeutic molecule to reverse age-related hypertension in humans. The reversal of age-related hypertension by restoring proteins that have undergone age-related modification is conceptually novel in the treatment of hypertension. Trx reverses age-related hypertension via maintaining vascular redox homeostasis, regenerating critical vasoregulatory proteins oxidized due to advancing age, and restoring native function of proteins that have undergone age-related modifications with loss-of function. Recent studies demonstrate that Trx is a promising molecule that may ameliorate or reverse age-related hypertension in older adults.

Thioredoxin reverses age-related hypertension by chronically improving vascular redox and restoring eNOS function.

The incidence of high blood pressure with advancing age is notably high, and it is an independent prognostic factor for the onset or progression of a variety of cardiovascular disorders. Although age-related hypertension is an established phenomenon, current treatments are only palliative but not curative. Thus, there is a critical need for a curative therapy against age-related hypertension, which could greatly decrease the incidence of cardiovascular disorders. We show that overexpression of human thioredoxin (TRX), a redox protein, in mice prevents age-related hypertension. Further, injection of recombinant human TRX (rhTRX) for three consecutive days reversed hypertension in aged wild-type mice, and this effect lasted for at least 20 days. Arteries of wild-type mice injected with rhTRX or mice with TRX overexpression exhibited decreased arterial stiffness, greater endothelium-dependent relaxation, increased nitric oxide production, and decreased superoxide anion (O2•-) generation compared to either saline-injected aged wild-type mice or mice with TRX deficiency. Our study demonstrates a potential translational role of rhTRX in reversing age-related hypertension with long-lasting efficacy.

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction.

Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.

Thioredoxin Activates MKK4-NFκB Pathway in a Redox-dependent Manner to Control Manganese Superoxide Dismutase Gene Expression in Endothelial Cells.

The mitogen-activated protein kinase kinase 4 (MKK4) is activated via phosphorylation of Ser-257 and Thr-261 by upstream MAP3Ks and activates JNK and p38 MAPKs in response to cellular stress. We show that thioredoxin (Trx), a cellular redox protein, activates MKK4 via Cys-246 and Cys-266 residues as mutation of these residues renders MKK4 insensitive to phosphorylation by MAP3Ks, TNFα, or Trx. MKK4 is activated in vitro by reduced Trx but not oxidized Trx in the absence of an upstream kinase, suggesting that autophosphorylation of this protein occurs due to reduction of Cys-246 and Cys-266 by Trx. Additionally, mutation of Cys-246 and Cys-266 resulted in loss of kinase activity suggesting that the redox state of Cys-246 and Cys-266 is a critical determinant of MKK4 activation. Trx induces manganese superoxide dismutase (MnSOD) gene transcription by activating MKK4 via redox control of Cys-246 and Cys-266, as mutation of these residues abrogates MKK4 activation and MnSOD expression. We further show that MKK4 activates NFκB for its binding to the MnSOD promoter, which leads to AP-1 dissociation followed by MnSOD transcription. Taken together, our studies show that the redox status of Cys-246 and Cys-266 in MKK4 controls its activities independent of MAP3K, demonstrating integration of the endothelial redox environment to MAPK signaling.

CD13 restricts TLR4 endocytic signal transduction in inflammation.

Dysregulation of the innate immune response underlies numerous pathological conditions. The TLR4 is the prototypical sensor of infection or injury that orchestrates the innate response via sequential activation of both cell surface and endocytic signaling pathways that trigger distinct downstream consequences. CD14 binds and delivers LPS to TLR4 and has been identified as a positive regulator of TLR4 signal transduction. It is logical that negative regulators of this process also exist to maintain the critical balance required for fighting infection, healing damaged tissue, and resolving inflammation. We showed that CD13 negatively modulates receptor-mediated Ag uptake in dendritic cells to control T cell activation in adaptive immunity. In this study, we report that myeloid CD13 governs internalization of TLR4 and subsequent innate signaling cascades, activating IRF-3 independently of CD14. CD13 is cointernalized with TLR4, CD14, and dynamin into Rab5(+) early endosomes upon LPS treatment. Importantly, in response to TLR4 ligands HMGB1 and LPS, p-IRF-3 activation and transcription of its target genes are enhanced in CD13(KO) dendritic cells, whereas TLR4 surface signaling remains unaffected, resulting in a skewed inflammatory response. This finding is physiologically relevant as ischemic injury in vivo provoked identical TLR4 responses. Finally, CD13(KO) mice showed significantly enhanced IFNß-mediated signal transduction via JAK-STAT, escalating inducible NO synthase transcription levels and promoting accumulation of oxidative stress mediators and tissue injury. Mechanistically, inflammatory activation of macrophages upregulates CD13 expression and CD13 and TLR4 coimmunoprecipitate. Therefore, CD13 negatively regulates TLR4 signaling, thereby balancing the innate response by maintaining the inflammatory equilibrium critical to innate immune regulation.

Involvement of inducible nitric oxide synthase and dimethyl arginine dimethylaminohydrolase in Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension.

Chronic administration of Nω-nitro-L-arginine methyl ester (L-NAME) in rats is a chemical method to study the induction and progression of nitric oxide (NO) deficiency-induced endothelial dysfunction. Male Wistar rats received L-NAME (50 mg/kg/day in drinking water) or no drug for 6 weeks. Mean arterial pressure (MAP) was measured on Day 43 by carotid artery cannulation. Plasma interleukin 1ß (IL-1ß) level was measured by enzyme-linked immunosorbent assay. Aorta and carotid artery were isolated for determination of basal nitrite, cGMP production, soluble guanylylcyclase (sGC) activity, phosphodiesterase-5 (PDE5) activity, and dimethylarginine dimethylaminohydrolase (DDAH) activity. mRNA expression studies were done by real time-polymerase chain reaction. L-NAME induced an increase in MAP and plasma IL-1ß. The treatment had varied effect on endothelial nitric oxide synthase (eNOS), sGC, and PDE5 but showed an increase in inducible NOS (iNOS) mRNA expression and plasma asymmetric dimethyl arginine levels. Basal nitrite, cGMP levels, sGC activity, and DDAH activity were significantly decreased in the tissues. Brief incubation of tissues in vitro with 1400 W, a specific iNOS blocker, partially reversed sGC activity, and cGMP levels. The results of this study showed that L-NAME-mediated inhibition of eNOS is only partially responsible for the vascular pathology observed in this model. Secondary effects that include an increase in iNOS and a decrease in DDAH activity are likely to be the causative factors for the progression of vascular dysfunction.

Molecular mechanisms regulating CD13-mediated adhesion.

CD13/Aminopeptidase N is a transmembrane metalloproteinase that is expressed in many tissues where it regulates various cellular functions. In inflammation, CD13 is expressed on myeloid cells, is up-regulated on endothelial cells at sites of inflammation and mediates monocyte/endothelial adhesion by homotypic interactions. In animal models the lack of CD13 alters the profiles of infiltrating inflammatory cells at sites of ischaemic injury. Here, we found that CD13 expression is enriched specifically on the pro-inflammatory subset of monocytes, suggesting that CD13 may regulate trafficking and function of specific subsets of immune cells. To further dissect the mechanisms regulating CD13-dependent trafficking we used the murine model of thioglycollate-induced sterile peritonitis. Peritoneal monocytes, macrophages and dendritic cells were significantly decreased in inflammatory exudates from global CD13(KO) animals when compared with wild-type controls. Furthermore, adoptive transfer of wild-type and CD13(KO) primary myeloid cells, or wild-type myeloid cells pre-treated with CD13-blocking antibodies into thioglycollate-challenged wild-type recipients demonstrated fewer CD13(KO) or treated cells in the lavage, suggesting that CD13 expression confers a competitive advantage in trafficking. Similarly, both wild-type and CD13(KO) cells were reduced in infiltrates in CD13(KO) recipients, confirming that both monocytic and endothelial CD13 contribute to trafficking. Finally, murine monocyte cell lines expressing mouse/human chimeric CD13 molecules demonstrated that the C-terminal domain of the protein mediates CD13 adhesion. Therefore, this work verifies that the altered inflammatory trafficking in CD13(KO) mice is the result of aberrant myeloid cell subset trafficking and further defines the molecular mechanisms underlying this regulation.

CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle.

Mesenchymal stem cells (MSCs) are multipotent, tissue-resident cells that can facilitate tissue regeneration and thus, show great promise as potential therapeutic agents. Functional MSCs have been isolated and characterized from a wide array of adult tissues and are universally identified by the shared expression of a core panel of MSCs markers. One of these markers is the multifunctional cell surface peptidase CD13 that has been shown to be expressed on human and murine MSCs from many tissues. To investigate whether this universal expression indicates a functional role for CD13 in MSC biology we isolated, expanded and characterized MSCs from bone marrow of wild type (WT) and CD13(KO) mice. Characterization of these cells demonstrated that both WT and CD13(KO) MSCs expressed the full complement of MSC markers (CD29, CD44, CD49e, CD105, Sca1), showed comparable proliferation rates and were capable of differentiating toward the adipogenic and osteogenic lineages. However, MSCs lacking CD13 were unable to differentiate into vascular cells, consistent with our previous characterization of CD13 as an angiogenic regulator. Compared to WT MSCs, adhesion and migration on various extracellular matrices of CD13(KO) MSCs were significantly impaired, which correlated with decreased phospho-FAK levels and cytoskeletal alterations. Crosslinking human MSCs with activating CD13 antibodies increased cell adhesion to endothelial monolayers and induced FAK activation in a time dependent manner. In agreement with these in vitro data, intramuscular injection of CD13(KO) MSCs in a model of severe ischemic limb injury resulted in significantly poorer perfusion, decreased ambulation, increased necrosis and impaired vascularization compared to those receiving WT MSCs. This study suggests that CD13 regulates FAK activation to promote MSC adhesion and migration, thus, contributing to MSC-mediated tissue repair. CD13 may present a viable target to enhance the efficacy of mesenchymal stem cell therapies.

CD13 regulates anchorage and differentiation of the skeletal muscle satellite stem cell population in ischemic injury.

CD13 is a multifunctional cell surface molecule that regulates inflammatory and angiogenic mechanisms in vitro, but its contribution to these processes in vivo or potential roles in stem cell biology remains unexplored. We investigated the impact of loss of CD13 on a model of ischemic skeletal muscle injury that involves angiogenesis, inflammation, and stem cell mobilization. Consistent with its role as an inflammatory adhesion molecule, lack of CD13 altered myeloid trafficking in the injured muscle, resulting in cytokine profiles skewed toward a prohealing environment. Despite this healing-favorable context, CD13(KO) animals showed significantly impaired limb perfusion with increased necrosis, fibrosis, and lipid accumulation. Capillary density was correspondingly decreased, implicating CD13 in skeletal muscle angiogenesis. The number of CD45-/Sca1-/α7-integrin+/ß1-integrin+ satellite cells was markedly diminished in injured CD13(KO) muscles and adhesion of isolated CD13(KO) satellite cells was impaired while their differentiation was accelerated. Bone marrow transplantation studies showed contributions from both host and donor cells to wound healing. Importantly, CD13 was coexpressed with Pax7 on isolated muscle-resident satellite cells. Finally, phosphorylated-focal adhesion kinase and ERK levels were reduced in injured CD13(KO) muscles, consistent with CD13 regulating satellite cell adhesion, potentially contributing to the maintenance and renewal of the satellite stem cell pool and facilitating skeletal muscle regeneration.

Tyrosine phosphorylation of CD13 regulates inflammatory cell-cell adhesion and monocyte trafficking.

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

CD13 is essential for inflammatory trafficking and infarct healing following permanent coronary artery occlusion in mice.

AIMS: To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. METHODS AND RESULTS: Seven days post-permanent ligation, hearts from CD13 knockout (CD13(KO)) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13(KO) infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13(KO) mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13(KO) infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13(KO) infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. CONCLUSION: In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

Novel interactions between NFATc1 (Nuclear Factor of Activated T cells c1) and STAT-3 (Signal Transducer and Activator of Transcription-3) mediate G protein-coupled receptor agonist, thrombin-induced biphasic expression of cyclin D1, with first phase influencing cell migration and second phase directing cell proliferation.

Thrombin, a G protein-coupled receptor agonist, induced a biphasic expression of cyclin D1 in primary vascular smooth muscle cells. Although both phases of cyclin D1 expression require binding of the newly identified cooperative complex, NFATc1·STAT-3, to its promoter, the second phase, which is more robust, depends on NFATc1-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT-3. In addition, STAT-3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires NFATc1-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT-3 by overexpression of acetylation null STAT-3 mutant led to the loss of the late phase of cyclin D1 expression. EMSA analysis and reporter gene assays revealed that NFATc1·STAT-3 complex binding to the cyclin D1 promoter led to an enhanceosome formation and facilitated cyclin D1 expression. In the early phase of its expression, cyclin D1 is localized mostly in the cytoplasm and influenced cell migration. However, during the late and robust phase of its expression, cyclin D1 is translocated to the nucleus and directed cell proliferation. Together, these results demonstrate for the first time that the dual function of cyclin D1 in cell migration and proliferation is temperospatially separated by its biphasic expression, which is mediated by cooperative interactions between NFATc1 and STAT-3.

CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake.

Dendritic cell (DC) Ag cross-presentation is generally associated with immune responses to tumors and viral Ags, and enhancement of this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8(+) murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA Ag, although development, maturation, and Ag processing and presentation of DCs are normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of Ags such as OVA and transferrin but not fluid-phase or phagocytic Ag uptake. CD13 and Ag are cointernalized in DCs, but CD13 did not coimmunoprecipitate with Ag receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs, and blocking of these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance Ag uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.

15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires Src-mediated Egr-1-dependent rapid induction of FGF-2 expression.

To understand the mechanisms underlying 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE]-induced angiogenesis, we studied the role of Egr-1. 15(S)-HETE induced Egr-1 expression in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Blockade of Egr-1 via forced expression of its dominant-negative mutant attenuated 15(S)-HETE-induced HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. 15(S)-HETE-induced Egr-1 expression requires Src activation. In addition, adenovirus-mediated expression of dominant-negative mutant of Src blocked 15(S)-HETE's effects on migration and tube formation of HDMVECs and Matrigel plug angiogenesis. 15(S)-HETE induced fibroblast growth factor-2 (FGF-2) expression rapidly via Src-mediated production of Egr-1. Cloning and mutational analysis of FGF-2 promoter revealed that Egr-1 binding site proximal to transcription start site is required for 15(S)-HETE-induced FGF-2 expression. Neutralizing antibody-mediated suppression of FGF-2 function also attenuated the effects of 15(S)-HETE on HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. Furthermore, in contrast to wild-type mice, 12/15-LOX(-/-) mice exhibited decreased Matrigel plug angiogenesis in response to AA, which was rescued by 15(S)-HETE. On the basis of these observations, we conclude that 15(S)-HETE-induced angiogenesis requires Src-mediated Egr-1-dependent rapid induction of FGF-2. These findings may suggest that 15(S)-HETE could be a potential endogenous regulator of pathologic angiogenesis associated with atherosclerosis and restenosis.

Essential role of nitric oxide in sepsis-induced impairment of endothelium-derived hyperpolarizing factor-mediated relaxation in rat pulmonary artery.

Both endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are important vasodilators in pulmonary circulation. Sepsis is known to impair endothelium-dependent dilation in the pulmonary vasculature, but the mechanisms are incompletely understood. We have examined the relative contribution of EDHF/NO to the attenuated endothelium-dependent relaxation of pulmonary artery in sepsis, and the role of inducible nitric oxide synthase (iNOS)-derived NO in this mechanism. Sepsis was induced in male adult Wistar rats by caecal ligation and puncture. At 18h after surgery, left and right branches of pulmonary arteries were isolated for tension recording, NO/cyclic guanosine monophosphate (cGMP) measurements, mRNA and protein expressions. Despite a marked decrease in the arterial endothelial nitric oxide synthase (eNOS) mRNA and phosphorylated-eNOS (p-eNOS) protein expressions in sepsis, endothelium-dependent relaxation to acetylcholine (ACh) mediated by NO, acetylcholine-stimulated NO release and tissue cGMP levels were moderately inhibited. Sepsis however abolished the N(G)-Nitro-l-arginine methyl ester (L-NAME)/indomethacin-resistant arterial relaxation (EDHF response) to acetylcholine in this vessel. In vitro treatment of the arterial rings from septic rats with 1400W, a selective inhibitor of iNOS restored the EDHF response, but had no effect on the acetylcholine-induced relaxation mediated by endothelial NO. The functional role of iNOS-derived NO in impairing EDHF-mediated relaxation was coincident with an increased basal NO production, iNOS mRNA and protein expressions in the rat pulmonary artery. In conclusion, the loss of the EDHF response may be primarily responsible for the endothelial dysfunction in sepsis, and its restoration by a selective iNOS inhibitor may improve pulmonary vasodilation.