TRIM13 reduces cholesterol efflux and increases oxidized LDL uptake leading to foam cell formation and atherosclerosis.

Impaired cholesterol efflux and/or uptake can influence arterial lipid accumulation leading to atherosclerosis. Here, we report that tripartite motif-containing protein 13 (TRIM13), a RING-type E3 ubiquitin ligase, plays a role in arterial lipid accumulation leading to atherosclerosis. Using molecular approaches and KO mouse model, we found that TRIM13 expression was induced both in the aorta and peritoneal macrophages (pMφ) of ApoE-/- mice in response to Western diet (WD) in vivo. Furthermore, proatherogenic cytokine interleukin-1ß also induced TRIM13 expression both in pMφ and vascular smooth muscle cells. Furthermore, we found that TRIM13 via ubiquitination and degradation of liver X receptor (LXR)α/ß downregulates the expression of their target genes ABCA1/G1 and thereby inhibits cholesterol efflux. In addition, TRIM13 by ubiquitinating and degrading suppressor of cytokine signaling 1/3 (SOCS1/3) mediates signal transducer and activator of transcription 1 (STAT1) activation, CD36 expression, and foam cell formation. In line with these observations, genetic deletion of TRIM13 by rescuing cholesterol efflux and inhibiting foam cell formation protects against diet-induced atherosclerosis. We also found that while TRIM13 and CD36 levels were increased, LXRα/ß, ABCA1/G1, and SOCS3 levels were decreased both in Mφ and smooth muscle cells of stenotic human coronary arteries as compared to nonstenotic arteries. More intriguingly, the expression levels of TRIM13 and its downstream signaling molecules were correlated with the severity of stenotic lesions. Together, these observations reveal for the first time that TRIM13 plays a crucial role in diet-induced atherosclerosis, and that it could be a potential drug target against this vascular lesion.

Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular Leakage.

BACKGROUND: Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels. METHODS: A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage. RESULTS: We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy-induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy-induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy-induced retinal neovascularization and vascular leakage, respectively. CONCLUSIONS: The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.

Blockade of CD47 function attenuates restenosis by promoting smooth muscle cell efferocytosis and inhibiting their migration and proliferation.

Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cß3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin ß3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia.

Novel Role of Prereplication Complex Component Cell Division Cycle 6 in Retinal Neovascularization.

BACKGROUND: The major aim of this study is to investigate whether CDC6 (cell division cycle 6), a replication origin recognition complex component, plays a role in retinal neovascularization, and if so, to explore the underlying mechanisms. METHODS: In this study, we used a variety of approaches including cellular and moleculer biological methodologies as well as global and tissue-specific knockout mice in combination with an oxygen-induced retinopathy model to study the role of CDC6 in retinal neovascularization. RESULTS: VEGFA (vascular endothelial growth factor A)-induced CDC6 expression in a time-dependent manner in human retinal microvascular endothelial cells. In addition, VEGFA-induced CDC6 expression was dependent on PLCß3 (phospholipase Cß3)-mediated NFATc1 (nuclear factor of activated T cells c1) activation. Furthermore, while siRNA-mediated depletion of PLCß3, NFATc1, or CDC6 levels blunted VEGFA-induced human retinal microvascular endothelial cell angiogenic events such as proliferation, migration, sprouting, and tube formation, CDC6 overexpression rescued these effects in NFATc1-deficient mouse retinal microvascular endothelial cells. In accordance with these observations, global knockdown of PLCß3 or endothelial cell-specific deletion of NFATc1 or siRNA-mediated depletion of CDC6 levels substantially inhibited oxygen-induced retinopathy-induced retinal sprouting and neovascularization. In addition, retroviral-mediated overexpression of CDC6 rescued oxygen-induced retinopathy-induced retinal neovascularization from inhibition in PLCß3 knockout mice and in endothelial cell-specific NFATc1-deficient mice. CONCLUSIONS: The above observations clearly reveal that PLCß3-mediated NFATc1 activation-dependent CDC6 expression plays a crucial role in VEGFA/oxygen-induced retinopathy-induced retinal neovascularization.

Myristoylation of LMCD1 Leads to Its Species-Specific Derepression of E2F1 and NFATc1 in the Modulation of CDC6 and IL-33 Expression During Development of Vascular Lesions.

OBJECTIVE: In view of our previous observations on differential expression of LMCD1 (LIM and cysteine-rich domains 1) in human versus rodents, we asked the question whether LMCD1 plays a species-specific role in the development of vascular lesions. Approach and Results: A combination of genetic, molecular, cellular, and disease models were used to test species-specific role of LMCD1 in the pathogenesis of vascular lesions. Here, we report species-specific regulation of LMCD1 expression in mediating vascular smooth muscle cell proliferation and migration during vascular wall remodeling in humans versus mice. Thrombin induced LMCD1 expression in human aortic smooth muscle cells but not mouse aortic smooth muscle cells via activation of Par1 (protease-activated receptor 1)-Gαq/11 (Gα protein q/11)-PLCß3 (phospholipase Cß3)-NFATc1 (nuclear factor of activated T cells 1) signaling. Furthermore, although LMCD1 mediates thrombin-induced proliferation and migration of both human aortic smooth muscle cells and mouse aortic smooth muscle cells via influencing E2F1 (E2F transcription factor 1)-mediated CDC6 (cell division cycle 6) expression and NFATc1-mediated IL (interleukin)-33 expression, respectively, in humans, it acts as an activator, and in mice, it acts as a repressor of these transcriptional factors. Interestingly, LMCD1 repressor activity was nullified by N-myristoyltransferase 2-mediated myristoylation in mouse. Besides, we found increased expression of LMCD1 in human stenotic arteries as compared to nonstenotic arteries. On the other hand, LMCD1 expression was decreased in neointimal lesions of mouse injured arteries as compared to noninjured arteries. CONCLUSIONS: Together, these observations reveal that LMCD1 acts as an activator and repressor of E2F1 and NFATc1 in humans and mice, respectively, in the induction of CDC6 and IL-33 expression during development of vascular lesions. Based on these findings, LMCD could be a potential target for drug development against restenosis and atherosclerosis in humans.

NFATc1-E2F1-LMCD1-Mediated IL-33 Expression by Thrombin Is Required for Injury-Induced Neointima Formation.

Objective- IL (interleukin)-33 has been shown to play a role in endothelial dysfunction, but its role in atherosclerosis is controversial. Therefore, the purpose of this study is to examine its role in vascular wall remodeling following injury. Approach and Results- Thrombin induced IL-33 expression in a time-dependent manner in human aortic smooth muscle cells and inhibition of its activity by its neutralizing antibody suppressed thrombin induced human aortic smooth muscle cell migration but not DNA synthesis. In exploring the mechanisms, we found that Par1 (protease-activated receptor 1), Gαq/11 (Gα protein q/11), PLCß3 (phospholipase Cß3), NFATc1 (nuclear factor of activated T cells), E2F1 (E2F transcription factor 1), and LMCD1 (LIM and cysteine-rich domains protein 1) are involved in thrombin-induced IL-33 expression and migration. Furthermore, we identified an NFAT-binding site at -100 nt that mediates thrombin-induced IL-33 promoter activity. Interestingly, we observed that NFATc1, E2F1, and LMCD1 bind to NFAT site in response to thrombin and found that LMCD1, while alone has no significant effect, enhanced either NFATc1 or E2F1-dependent IL-33 promoter activity. In addition, we found that guidewire injury induces IL-33 expression in SMC and its neutralizing antibodies substantially reduce SMC migration and neointimal growth in vivo. Increased expression of IL-33 was also observed in human atherosclerotic lesions as compared to arteries without any lesions. Conclusions- The above findings reveal for the first time that thrombin-induced human aortic smooth muscle cell migration and injury-induced neointimal growth require IL-33 expression. In addition, thrombin-induced IL-33 expression requires LMCD1 enhanced combinatorial activation of NFATc1 and E2F1.

Protease-activated receptor 1 inhibits cholesterol efflux and promotes atherogenesis via cullin 3-mediated degradation of the ABCA1 transporter.

Although signaling of thrombin via its receptor protease-activated receptor 1 (Par1) is known to occur in atherothrombosis, its link to the actual pathogenesis of this condition is less clear. To better understand the role of thrombin-Par1 signaling in atherosclerosis, here we have studied their effects on cellular cholesterol efflux in mice. We found that by activating Par1 and cullin 3-mediated ubiquitination and degradation of ABC subfamily A member 1 (ABCA1), thrombin inhibits cholesterol efflux in both murine macrophages and smooth muscle cells. Moreover, disruption of the Par1 gene rescued ABCA1 from Western diet-induced ubiquitination and degradation and restored cholesterol efflux in apolipoprotein E-deficient (ApoE-/-) mice. Similarly, the Par1 deletion diminished diet-induced atherosclerotic lesions in the ApoE-/- mice. These observations for the first time indicate a role for thrombin-Par1 signaling in the pathogenesis of diet-induced atherosclerosis. We identify cullin 3 as a cullin-RING ubiquitin E3 ligase that mediates ABCA1 ubiquitination and degradation and thereby inhibits cholesterol efflux. Furthermore, compared with peripheral blood mononuclear cells (PBMCs) from ApoE-/- mice, the PBMCs from ApoE-/-:Par1-/- mice exhibited decreased trafficking to inflamed arteries of Western diet-fed ApoE-/- mice. This finding suggested that besides inhibiting cholesterol efflux, thrombin-Par1 signaling also plays a role in the recruitment of leukocytes during diet-induced atherogenesis. Based on these findings, we conclude that thrombin-Par1 signaling appears to contribute to the pathogenesis of atherosclerosis by impairing cholesterol efflux from cells and by recruiting leukocytes to arteries.

LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis.

Restenosis arises after vascular injury and is characterized by arterial wall thickening and decreased arterial lumen space. Vascular injury induces the production of thrombin, which in addition to its role in blood clotting acts as a mitogenic and chemotactic factor. In exploring the molecular mechanisms underlying restenosis, here we identified LMCD1 (LIM and cysteine-rich domains 1) as a gene highly responsive to thrombin in human aortic smooth muscle cells (HASMCs). Of note, LMCD1 depletion inhibited proliferation of human but not murine vascular smooth muscle cells. We also found that by physically interacting with E2F transcription factor 1, LMCD1 mediates thrombin-induced expression of the CDC6 (cell division cycle 6) gene in the stimulation of HASMC proliferation. Thrombin-induced LMCD1 and CDC6 expression exhibited a requirement for protease-activated receptor 1-mediated Gαq/11-dependent activation of phospholipase C ß3. Moreover, the expression of LMCD1 was highly induced in smooth muscle cells located at human atherosclerotic lesions and correlated with CDC6 expression and that of the proliferation marker Ki67. Furthermore, the LMCD1- and SMCαactin-positive cells had higher cholesterol levels in the atherosclerotic lesions. In conclusion, these findings indicate that by acting as a co-activator with E2F transcription factor 1 in CDC6 expression, LMCD1 stimulates HASMC proliferation and thereby promotes human atherogenesis, suggesting an involvement of LMCD1 in restenosis.

Protein Kinase Cθ Via Activating Transcription Factor 2-Mediated CD36 Expression and Foam Cell Formation of Ly6Chi Cells Contributes to Atherosclerosis.

BACKGROUND: Although the role of thrombin in atherothrombosis is well studied, its role in the pathogenesis of diet-induced atherosclerosis is not known. METHODS: Using a mouse model of diet-induced atherosclerosis and molecular biological approaches, here we have explored the role of thrombin and its G protein-coupled receptor signaling in diet-induced atherosclerosis. RESULTS: In exploring the role of G protein-coupled receptor signaling in atherogenesis, we found that thrombin triggers foam cell formation via inducing CD36 expression, and these events require Par1-mediated Gα12-Pyk2-Gab1-protein kinase C (PKC)θ-dependent ATF2 activation. Genetic deletion of PKCθ in apolipoprotein E (ApoE)-/- mice reduced Western diet-induced plaque formation. Furthermore, thrombin induced Pyk2, Gab1, PKCθ, and ATF2 phosphorylation, CD36 expression, and foam cell formation in peritoneal macrophages of ApoE-/- mice. In contrast, thrombin only stimulated Pyk2 and Gab1 but not ATF2 phosphorylation or its target gene CD36 expression in the peritoneal macrophages of ApoE-/-:PKCθ-/- mice, and it had no effect on foam cell formation. In addition, the aortic root cross-sections of Western diet-fed ApoE-/- mice showed increased Pyk2, Gab1, PKCθ, and ATF2 phosphorylation and CD36 expression as compared with ApoE-/-:PKCθ-/- mice. Furthermore, although the monocytes from peripheral blood and the aorta of Western diet-fed ApoE-/- mice were found to contain more of Ly6Chi cells than Ly6Clo cells, the monocytes from Western diet-fed ApoE-/-:PKCθ-/- mice were found to contain more Ly6Clo cells than Ly6Chi cells. It is interesting to note that the Ly6Chi cells showed higher CD36 expression with enhanced capacity to form foam cells as compared with Ly6Clo cells. CONCLUSIONS: These findings reveal for the first time that thrombin-mediated Par1-Gα12 signaling via targeting Pyk2-Gab1-PKCθ-ATF2-dependent CD36 expression might be playing a crucial role in diet-induced atherogenesis.

p115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine-induced vascular smooth muscle cell migration and proliferation.

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-Gi/o-Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling.

Heterodimers of the transcriptional factors NFATc3 and FosB mediate tissue factor expression for 15(S)-hydroxyeicosatetraenoic acid-induced monocyte trafficking.

Tissue factor (TF) is expressed in vascular and nonvascular tissues and functions in several pathways, including embryonic development, inflammation, and cell migration. Many risk factors for atherosclerosis, including hypertension, diabetes, obesity, and smoking, increase TF expression. To better understand the TF-related mechanisms in atherosclerosis, here we investigated the role of 12/15-lipoxygenase (12/15-LOX) in TF expression. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major product of human 15-LOXs 1 and 2, induced TF expression and activity in a time-dependent manner in the human monocytic cell line THP1. Moreover, TF suppression with neutralizing antibodies blocked 15(S)-HETE-induced monocyte migration. We also found that NADPH- and xanthine oxidase-dependent reactive oxygen species (ROS) production, calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation, and interactions between nuclear factor of activated T cells 3 (NFATc3) and FosB proto-oncogene, AP-1 transcription factor subunit (FosB) are involved in 15(S)-HETE-induced TF expression. Interestingly, NFATc3 first induced the expression of its interaction partner FosB before forming the heterodimeric NFATc3-FosB transcription factor complex, which bound the proximal AP-1 site in the TF gene promoter and activated TF expression. We also observed that macrophages from 12/15-LOX-/- mice exhibit diminished migratory response to monocyte chemotactic protein 1 (MCP-1) and lipopolysaccharide compared with WT mouse macrophages. Similarly, compared with WT macrophages, monocytes from 12/15-LOX-/- mice displayed diminished trafficking, which was rescued by prior treatment with 12(S)-HETE, in a peritonitis model. These observations indicate that 15(S)-HETE-induced monocyte/macrophage migration and trafficking require ROS-mediated CaMKIV activation leading to formation of NFATc3 and FosB heterodimer, which binds and activates the TF promoter.

A new role for cofilin in retinal neovascularization.

Pak1 plays an important role in several cellular processes, including cell migration, but its role in pathological angiogenesis is not known. Here, we have determined its role in pathological retinal angiogenesis using an oxygen-induced retinopathy (OIR) model. VEGFA induced phosphorylation of Pak1 and its effector cofilin in a manner that was dependent on time as well as p38MAPKß (also known as MAPK11) in human retinal microvascular endothelial cells (HRMVECs). Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVEC F-actin stress fiber formation, migration, proliferation, sprouting and tube formation. In accordance with these observations, hypoxia induced Pak1 and cofilin phosphorylation with p38MAPKß being downstream to Pak1 and upstream to cofilin in mouse retina. Furthermore, Pak1 deficiency abolished hypoxia-induced p38MAPKß and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, small interfering RNA (siRNA)-mediated downregulation of p38MAPKß or cofilin levels in the wild-type mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Taken together, these observations suggest that, although the p38MAPKß-Pak1-cofilin axis is required for HRMVEC migration, proliferation, sprouting and tubulogenesis, Pak1-p38MAPKß-cofilin signaling is also essential for hypoxia-induced mouse retinal endothelial cell proliferation, tip cell formation and neovascularization.

Reactive Oxygen Species (ROS) Mediate p300-dependent STAT1 Protein Interaction with Peroxisome Proliferator-activated Receptor (PPAR)-γ in CD36 Protein Expression and Foam Cell Formation.

Previously, we have demonstrated that 15(S)-hydroxyeicosatetranoic acid (15(S)-HETE) induces CD36 expression involving STAT1. Many studies have shown that peroxisome proliferator-activated receptor (PPAR)-γ mediates CD36 expression. Therefore, we asked the question whether these transcriptional factors interact with each other in the regulation of CD36 expression by 15(S)-HETE. Here, we show that STAT1 interacts with PPARγ in the induction of CD36 expression and foam cell formation by 15(S)-HETE. In addition, using molecular biological approaches such as EMSA, supershift EMSA, ChIP, re-ChIP, and promoter-reporter gene assays, we demonstrate that the STAT1 and PPARγ complex binds to the STAT-binding site at -107 nucleotides in the CD36 promoter and enhances its activity. Furthermore, the interaction of STAT1 with PPARγ depends on STAT1 acetylation, which is mediated by p300. In addition, our findings show that reactive oxygen species-dependent Syk and Pyk2 stimulation is required for p300 tyrosine phosphorylation and activation. Together, these results demonstrate that an interaction between STAT1, p300, and peroxisome proliferator-activated receptor-γ is required for 15(S)-HETE-induced CD36 expression, oxidized low density lipoprotein uptake, and foam cell formation, critical events underlying the pathogenesis of atherosclerosis.

12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction.

To understand the mechanisms of 15(S)-HETE-induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)-HETE induced junction adhesion molecule A (JamA) phosphorylation on Y164, Y218, and Y280 involving XO-mediated reactive oxygen species production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction (TJ) disruption, increased vascular permeability, and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appears to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high-fat diet (HFD) feeding, WT, but not 12/15-lipoxygenase (LO)(-/-), mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by allopurinol. These observations provide novel insights on the role of XO in 12/15-LO-induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.

Vascular endothelial tight junctions and barrier function are disrupted by 15(S)-hydroxyeicosatetraenoic acid partly via protein kinase C ε-mediated zona occludens-1 phosphorylation at threonine 770/772.

Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCε-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCε and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO(-/-) mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction.