Thioredoxin Prevents Loss of UCP2 in Hyperoxia via MKK4-p38 MAPK-PGC1α Signaling and Limits Oxygen Toxicity.

Administration of high concentrations of oxygen (hyperoxia) is one of few available options to treat acute hypoxemia-related respiratory failure, as seen in the current coronavirus disease (COVID-19) pandemic. Although hyperoxia can cause acute lung injury through increased production of superoxide anion (O2•-), the choice of high-concentration oxygen administration has become a necessity in critical care. The objective of this study was to test the hypothesis that UCP2 (uncoupling protein 2) has a major function of reducing O2•- generation in the lung in ambient air or in hyperoxia. Lung epithelial cells and wild-type; UCP2-/-; or transgenic, hTrx overexpression-bearing mice (Trx-Tg) were exposed to hyperoxia and O2•- generation was measured by using electron paramagnetic resonance, and lung injury was measured by using histopathologic analysis. UCP2 expression was analyzed by using RT-PCR analysis, Western blotting analysis, and RNA interference. The signal transduction pathways leading to loss of UCP2 expression were analyzed by using IP, phosphoprotein analysis, and specific inhibitors. UCP2 mRNA and protein expression were acutely decreased in hyperoxia, and these decreases were associated with a significant increase in O2•- production in the lung. Treatment of cells with rhTrx (recombinant human thioredoxin) or exposure of Trx-Tg mice prevented the loss of UCP2 protein and decreased O2•- generation in the lung. Trx is also required to maintain UCP2 expression in normoxia. Loss of UCP2 in UCP2-/- mice accentuated lung injury in hyperoxia. Trx activates the MKK4-p38MAPK (p38 mitogen-activated protein kinase)-PGC1α (PPARγ [peroxisome proliferator-activated receptor γ] coactivator 1α) pathway, leading to rescue of UCP2 and decreased O2•- generation in hyperoxia. Loss of UCP2 in hyperoxia is a major mechanism of O2•- production in the lung in hyperoxia. rhTrx can protect against lung injury in hyperoxia due to rescue of the loss of UCP2.

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.

Nuclear factor of activated T cells c1 mediates p21-activated kinase 1 activation in the modulation of chemokine-induced human aortic smooth muscle cell F-actin stress fiber formation, migration, and proliferation and injury-induced vascular wall remodeling.

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.

12/15-Lipoxygenase mediates high-fat diet-induced endothelial tight junction disruption and monocyte transmigration: a new role for 15(S)-hydroxyeicosatetraenoic acid in endothelial cell dysfunction.

A convincing body of evidence suggests that 12/15-lipoxygenase (12/15-LO) plays a role in atherosclerosis. However, the mechanisms of its involvement in the pathogenesis of this disease are not clear. Therefore, the purpose of this study is to understand the mechanisms by which 12/15-LO mediates endothelial dysfunction. 15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 12/15-LO metabolite of arachidonic acid (AA), induced endothelial barrier permeability via Src and Pyk2-dependent zonula occluden (ZO)-2 tyrosine phosphorylation and its dissociation from the tight junction complexes. 15(S)-HETE also stimulated macrophage adhesion to the endothelial monolayer in Src and Pyk2-dependent manner. Ex vivo studies revealed that exposure of arteries from WT mice to AA or 15(S)-HETE led to Src-Pyk2-dependent ZO-2 tyrosine phosphorylation, tight junction disruption, and macrophage adhesion, whereas the arteries from 12/15-LO knock-out mice are protected from these effects of AA. Feeding WT mice with a high-fat diet induced the expression of 12/15-LO in the arteries leading to tight junction disruption and macrophage adhesion and deletion of the 12/15-LO gene disallowed these effects. Thus, the findings of this study provide the first evidence of the role of 12/15-LO and its AA metabolite, 15(S)-HETE, in high-fat diet-induced endothelial tight junction disruption and macrophage adhesion, the crucial events underlying the pathogenesis of atherosclerosis.

Both Kdr and Flt1 play a vital role in hypoxia-induced Src-PLD1-PKCγ-cPLA(2) activation and retinal neovascularization.

To understand the mechanisms of Src-PLD1-PKCγ-cPLA2 activation by vascular endothelial growth factor A (VEGFA), we studied the role of Kdr and Flt1. VEGFA, while having no effect on Flt1 phosphorylation, induced Kdr phosphorylation in human retinal microvascular endothelial cells (HRMVECs). Depletion of Kdr attenuated VEGFA-induced Src-PLD1-PKCγ-cPLA2 activation. Regardless of its phosphorylation state, downregulation of Flt1 also inhibited VEGFA-induced Src-PLD1-PKCγ-cPLA2 activation, but only modestly. In line with these findings, depletion of either Kdr or Flt1 suppressed VEGFA-induced DNA synthesis, migration, and tube formation, albeit more robustly with Kdr downregulation. Hypoxia induced tyrosine phosphorylation of Kdr and Flt1 in mouse retina, and depletion of Kdr or Flt1 blocked hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization. VEGFB induced Flt1 tyrosine phosphorylation and Src-PLD1-PKCγ-cPLA2 activation in HRMVECs. Hypoxia induced VEGFA and VEGFB expression in retina, and inhibition of their expression blocked hypoxia-induced Kdr and Flt1 activation, respectively. Furthermore, depletion of VEGFA or VEGFB attenuated hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization. These findings suggest that although VEGFA, through Kdr and Flt1, appears to be the major modulator of Src-PLD1-PKCγ-cPLA2 signaling in HRMVECs, facilitating their angiogenic events in vitro, both VEGFA and VEGFB mediate hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization via activation of Kdr and Flt1, respectively.

Protein kinase N1 is a novel substrate of NFATc1-mediated cyclin D1-CDK6 activity and modulates vascular smooth muscle cell division and migration leading to inward blood vessel wall remodeling.

Toward understanding the mechanisms of vascular wall remodeling, here we have studied the role of NFATc1 in MCP-1-induced human aortic smooth muscle cell (HASMC) growth and migration and injury-induced rat aortic wall remodeling. We have identified PKN1 as a novel downstream target of NFATc1-cyclin D1/CDK6 activity in mediating vascular wall remodeling following injury. MCP-1, a potent chemoattractant protein, besides enhancing HASMC motility, also induced its growth, and these effects require NFATc1-dependent cyclin D1 expression and CDK4/6 activity. In addition, MCP-1 induced PKN1 activation in a sustained and NFATc1-cyclin D1/CDK6-dependent manner. Furthermore, PKN1 activation is required for MCP-1-induced HASMC growth and migration. Balloon injury induced PKN1 activation in NFAT-dependent manner and pharmacological or dominant negative mutant-mediated blockade of PKN1 function or siRNA-mediated down-regulation of its levels substantially suppressed balloon injury-induced smooth muscle cell migration and proliferation resulting in reduced neointima formation. These novel findings suggest that PKN1 plays a critical role in vascular wall remodeling, and therefore, it could be a promising new target for the next generation of drugs for vascular diseases, particularly restenosis following angioplasty, stent implantation, or vein grafting.

Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury.

OBJECTIVE: To investigate the role of Pyk2, a proline-rich nonreceptor tyrosine kinase, in G protein-coupled receptor agonist, thrombin-induced human aortic smooth muscle cell growth and migration, and injury-induced vascular wall remodeling. METHODS AND RESULTS: Thrombin, a G protein-coupled receptor agonist, activated Pyk2 in a time-dependent manner and inhibition of its stimulation attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. Thrombin also activated Grb2-associated binder protein 1, p115 Rho guanine nucleotide exchange factor, Rac1, RhoA, and p21-activated kinase 1 (Pak1) and interference with stimulation of these molecules attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. In addition, adenovirus-mediated expression of dominant negative Pyk2 inhibited thrombin-induced Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA and Pak1 stimulation. Balloon injury also caused activation of Pyk2, Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA, and Pak1 in the carotid artery of rat, and these responses were sensitive to inhibition by the dominant negative Pyk2. Furthermore, inhibition of Pyk2 activation resulted in reduced recruitment of smooth muscle cells onto the luminal surface and their proliferation in the intimal region leading to suppression of neointima formation. CONCLUSIONS: Together, these results demonstrate for the first time that Pyk2 plays a crucial role in G protein-coupled receptor agonist thrombin-induced human aortic smooth muscle cell growth and migration, as well as balloon injury-induced neointima formation.

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.

12/15-Lipoxygenase gene knockout severely impairs ischemia-induced angiogenesis due to lack of Rac1 farnesylation.

To understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates Rac1 in the induction of angiogenesis, we studied the role of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and αPix. 15(S)-HETE stimulated Rac1 in a sustained manner in human dermal microvascular endothelial cells (HDMVECs). Simvastatin, a potent inhibitor of HMG-CoA reductase, suppressed 15(S)-HETE-induced Rac1 activation in HDMVECs affecting their migration and tube formation. 15(S)-HETE by inducing HMG-CoA reductase expression caused increased farnesylation and membrane translocation of Rac1 where it became activated by Src-dependent αPix stimulation. Mevalonate rescued 15(S)-HETE-induced Rac1 farnesylation and membrane translocation in HDMVECs and the migration and tube formation of these cells from inhibition by simvastatin. Down-regulation of αPix inhibited 15(S)-HETE-induced HDMVEC migration and tube formation. Hind-limb ischemia induced Rac1 farnesylation and activation leading to increased angiogenesis and these effects were blocked by simvastatin and rescued by mevalonate in WT mice. In contrast, hind-limb ischemia failed to induce Rac1 farnesylation and activation as well as angiogenic response in 12/15-Lox(-/-) mice. Activation of Src and αPix were also compromised at least to some extent in 12/15-Lox(-/-) mice compared with WT mice in response to hind-limb ischemia. Together, these findings demonstrate for the first time that HMG-CoA reductase plays a determinant role in 12/15-Lox-induced angiogenesis.

15-Lipoxygenase-1-enhanced Src-Janus kinase 2-signal transducer and activator of transcription 3 stimulation and monocyte chemoattractant protein-1 expression require redox-sensitive activation of epidermal growth factor receptor in vascular wall remodeling.

To understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates signal transducer and activator of transcription 3 (STAT3), we studied the role of epidermal growth factor receptor (EGFR). 15(S)-HETE stimulated tyrosine phosphorylation of EGFR in a time-dependent manner in vascular smooth muscle cells (VSMCs). Interference with EGFR activation blocked 15(S)-HETE-induced Src and STAT3 tyrosine phosphorylation, monocyte chemoattractant protein-1 (MCP-1) expression and VSMC migration. 15(S)-HETE also induced tyrosine phosphorylation of Janus kinase 2 (Jak2) in VSMCs, and its inhibition substantially reduced STAT3 phosphorylation, MCP-1 expression, and VSMC migration. In addition, Src formed a complex with EGFR and Jak2, and its inhibition completely blocked Jak2 and STAT3 phosphorylation, MCP-1 expression, and VSMC migration. 15(S)-HETE induced the production of H(2)O(2) via an NADPH oxidase-dependent manner and its scavengers, N-acetyl cysteine (NAC) and catalase suppressed 15(S)-HETE-stimulated EGFR, Src, Jak2, and STAT3 phosphorylation and MCP-1 expression. Balloon injury (BI) induced EGFR, Src, Jak2, and STAT3 phosphorylation, and inhibition of these signaling molecules attenuated BI-induced MCP-1 expression and smooth muscle cell migration from the medial to the luminal surface resulting in reduced neointima formation. In addition, inhibition of EGFR blocked BI-induced Src, Jak2, and STAT3 phosphorylation. Similarly, interference with Src activation suppressed BI-induced Jak2 and STAT3 phosphorylation. Furthermore, adenovirus-mediated expression of dnJak2 also blocked BI-induced STAT3 phosphorylation. Consistent with the effects of 15(S)-HETE on the activation of EGFR-Src-Jak2-STAT3 signaling in VSMCs in vitro, adenovirus-mediated expression of 15-lipoxygenase 1 (15-Lox1) enhanced BI-induced EGFR, Src, Jak2, and STAT3 phosphorylation leading to enhanced MCP-1 expression in vivo. Blockade of Src or Jak2 suppressed BI-induced 15-Lox1-enhanced STAT3 phosphorylation, MCP-1 expression, and neointima formation. In addition, whereas dominant negative Src blocked BI-induced 15-Lox1-enhanced Jak2 phosphorylation, dnJak2 had no effect on Src phosphorylation. Together, these observations demonstrate for the first time that the 15-Lox1-15(S)-HETE axis activates EGFR via redox-sensitive manner, which in turn mediates Src-Jak2-STAT3-dependent MCP-1 expression leading to vascular wall remodeling.

Activation of cytosolic phospholipase A2 downstream of the Src-phospholipase D1 (PLD1)-protein kinase C γ (PKCγ) signaling axis is required for hypoxia-induced pathological retinal angiogenesis.

In view of understanding the mechanisms of retinal neovascularization, we had reported previously that vascular endothelial growth factor (VEGF)-induced pathological retinal angiogenesis requires the activation of Src-PLD1-PKCγ signaling. In the present work, we have identified cytosolic phospholipase A(2) (cPLA(2)) as an effector molecule of Src-PLD1-PKCγ signaling in the mediation of VEGF-induced pathological retinal angiogenesis based on the following observations. VEGF induced cPLA(2) phosphorylation in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). VEGF also induced arachidonic acid (AA) release in a dose-, time-, and cPLA(2)-dependent manner. Depletion of cPLA(2) levels inhibited VEGF-induced HRMVEC DNA synthesis, migration, and tube formation. In addition, the exogenous addition of AA rescued VEGF-induced HRMVEC DNA synthesis, migration, and tube formation from inhibition by down-regulation of cPLA(2). Inhibition of Src, PLD1, or PKCγ attenuated VEGF-induced cPLA(2) phosphorylation and AA release. Consistent with these findings, hypoxia induced cPLA(2) phosphorylation and activity in VEGF-Src-PLD1-PKCγ-dependent manner in a mouse model of oxygen-induced retinopathy. In addition, siRNA-mediated down-regulation of cPLA(2) levels in the retina abrogated hypoxia-induced retinal endothelial cell proliferation and neovascularization. These observations suggest that cPLA(2)-dependent AA release is required for VEGF-induced Src-PLD1-PKCγ-mediated pathological retinal angiogenesis.

AP-1 (Fra-1/c-Jun)-mediated induction of expression of matrix metalloproteinase-2 is required for 15S-hydroxyeicosatetraenoic acid-induced angiogenesis.

To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.