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// Suowen Xu 1 , Bin Liu 1 , Meimei Yin 1 , Marina Koroleva 1 , Michael Mastrangelo 1 , Sara Ture 1 , Craig N. Morrell 1 , David X. Zhang 2 , Edward A. Fisher 3 and Zheng Gen Jin 1 1 Aab Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA 2 Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA 3 Department of Medicine, Division of Cardiology, and The Marc and Ruti Bell Program in Vascular Biology, New York University School of Medicine, New York, NY, USA Correspondence to: Zheng Gen Jin, email: // Keywords : AMPK, atherosclerosis, GSK1016790A, shear stress, TRPV4 Received : December 29, 2015 Accepted : April 29, 2016 Published : May 14, 2016 Abstract TRPV4 ion channel mediates vascular mechanosensitivity and vasodilation. Here, we sought to explore whether non-mechanical activation of TRPV4 could limit vascular inflammation and atherosclerosis. We found that GSK1016790A, a potent and specific small-molecule agonist of TRPV4, induces the phosphorylation and activation of eNOS partially through the AMPK pathway. Moreover, GSK1016790A inhibited TNF-α-induced monocyte adhesion to human endothelial cells. Mice given GSK1016790A showed increased phosphorylation of eNOS and AMPK in the aorta and decreased leukocyte adhesion to TNF-α-inflamed endothelium. Importantly, oral administration of GSK1016790A reduced atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet. Together, the present study suggests that pharmacological activation of TRPV4 may serve as a potential therapeutic approach to treat atherosclerosis.

Growth factors and their downstream receptor tyrosine kinases (RTKs) mediate a number of biological processes controlling cell function. Adaptor (docking) proteins, which consist exclusively of domains and motifs that mediate molecular interactions, link receptor activation to downstream effectors. Recent studies have revealed that Grb2-associated-binders (Gab) family members (including Gab1, Gab2, and Gab3), when phosphorylated on tyrosine residues, provide binding sites for multiple effector proteins, such as Src homology-2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) and phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, thereby playing important roles in transducing RTKs-mediated signals into pathways with diversified biological functions. Here, we provide an up-to-date overview on the domain structure and biological functions of Gab1, the most intensively studied Gab family protein, in growth factor signaling and biological functions, with a special focus on angiogenesis.

Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs.

Skeletal muscle responds to exercise by activation of signalling pathways that co-ordinate gene expression to sustain muscle performance. MEF2 (myocyte enhancer factor 2)-dependent transcriptional activation of MHC (myosin heavy chain) genes promotes the transformation from fast-twitch into slow-twitch fibres, with MEF2 activity being tightly regulated by interaction with class IIa HDACs (histone deacetylases). PKD (protein kinase D) is known to directly phosphorylate skeletal muscle class IIa HDACs, mediating their nuclear export and thus derepression of MEF2. In the present study, we report the generation of transgenic mice with inducible conditional expression of a dominant-negative PKD1kd (kinase-dead PKD1) protein in skeletal muscle to assess the role of PKD in muscle function. In control mice, long-term voluntary running experiments resulted in a switch from type IIb+IId/x to type IIa plantaris muscle fibres as measured by indirect immunofluorescence of MHCs isoforms. In mice expressing PKD1kd, this fibre type switch was significantly impaired. These mice exhibited altered muscle fibre composition and decreased running performance compared with control mice. Our findings thus indicate that PKD activity is essential for exercise-induced MEF2-dependent skeletal muscle remodelling in vivo.

VEGF receptor 2 (VEGFR2), a tyrosine kinase receptor, is activated by VEGF and fluid shear stress (FSS), and its downstream signaling is important in the regulation of endothelial functions, such as cell migration, endothelium-dependent relaxation, and angiogenesis. Cigarette smoke (CS) is known to cause oxidative/nitrosative stress, leading to modifications of tyrosine kinase receptors and impaired downstream signaling. We hypothesized that CS-induced oxidative/nitrosative stress impairs VEGF- and FSS-mediated VEGFR2 activation, leading to endothelial dysfunction. Human lung microvascular endothelial cells and human umbilical vein endothelial cells were treated with different concentrations of cigarette smoke extract (CSE) to investigate the VEGF- or FSS-mediated VEGFR2 phosphorylation and its downstream signaling involved in endothelial function. CSE treatment impaired both VEGF- and FSS-mediated VEGFR2 phosphorylation, resulting in impaired endothelial nitric oxide synthase (eNOS) phosphorylation by Akt. CS-derived reactive oxygen/nitrogen species react with VEGFR2, rendering VEGFR2 inactive for its downstream signaling. Pretreatment with nitric oxide scavenger (PTIO), reactive oxygen species scavengers (combination of SOD with catalase), and N-acetyl-l-cysteine, significantly attenuated the CSE-induced impairment of VEGF-mediated Akt and eNOS phosphorylation. These findings suggest that CSE-induced oxidative/nitrosative stress impairs VEGF- and FSS-mediated endothelial cell function and has important implications in the pathogenesis of CS-induced pulmonary and cardiovascular diseases associated with endothelial dysfunction. Antioxid. Redox Signal. 12, 1355–1369.

Metabolic syndrome accelerates the atherosclerotic process, and the earliest event of which is endothelial dysfunction. Ghrelin, a newly discovered gastric peptide, improves endothelial function and inhibits proatherogenic changes. In particular, low ghrelin concentration has been associated with several features of metabolic syndrome, including obesity, insulin resistance, and high blood pressure. However, the molecular mechanisms underlying ghrelin vascular actions remain largely unclear. Here, we showed that ghrelin activated endothelial nitric oxide (NO) synthase (eNOS) in cultured endothelial cells (ECs) and in intact vessels. Specifically, ghrelin rapidly induced phosphorylation of eNOS on an activation site and production of NO in human umbilical vein ECs and bovine aortic ECs. The eNOS phosphorylation was also observed in mouse aortas ex vivo perfused with ghrelin and in aortic tissues isolated from mice injected with ghrelin. Mechanistically, ghrelin stimulated AMP-activated protein kinase (AMPK) and Akt activation in cultured ECs and intact vessels. Inhibiting AMPK and Akt with their pharmacological inhibitors, small interference RNA and adenoviruses carried dominant-negative mutants, markedly attenuated ghrelin-induced eNOS activation, and NO production. Furthermore, ghrelin receptor/Gq protein/calcium-dependent pathway mediates activation of AMPK, Akt, and eNOS, and calmodulin-dependent kinase kinase is a potential convergent point to regulate Akt and AMPK activation in ghrelin signaling. Importantly, eNOS activation is critical for ghrelin inhibition of vascular inflammation. Together, both in vitro and in vivo data demonstrate a new role of ghrelin signaling for eNOS activation, and highlight the therapeutic potential for ghrelin to correct endothelial dysfunction associated with atherosclerotic vascular diseases and metabolic syndrome.

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cgamma-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.

Aims Vascular endothelial dysfunction and inflammation are hallmarks of atherosclerosis. Kruppel-like factor 2 (KLF2) is a key mediator of anti-inflammatory and anti-atherosclerotic properties of the endothelium. However, little is known of the molecular mechanisms for regulating KLF2 transcriptional activation. Methods and results Here, we found that histone deacetylase 5 (HDAC5) associates with KLF2 and represses KLF2 transcriptional activation. HDAC5 resided with KLF2 in the nuclei of human umbilical cord vein endothelial cells (HUVECs). Steady laminar flow attenuated the association of HDAC5 with KLF2 via stimulating HDAC5 phosphorylation-dependent nuclear export in HUVEC. We also mapped the KLF2–HDAC5-interacting domains and found that the N-terminal region of HDAC5 interacts with the C-terminal domain of KLF2. Chromatin immunoprecipitation and luciferase reporter assays showed that HDAC5 through a direct association with KLF2 suppressed KLF2 transcriptional activation. HDAC5 overexpression inhibited KLF2-dependent endothelial nitric oxide synthesis (eNOS) promoter activity in COS7 cell and gene expression in both HUVECs and bovine aortic endothelial cells (BAECs). Conversely, HDAC5 silencing enhanced KLF2 transcription and hence eNOS expression in HUVEC. Moreover, we observed that the level of eNOS protein in the thoracic aorta isolated from HDAC5 knockout mice was higher, whereas expression of pro-inflammatory vascular cell adhesion molecule 1 was lower, compared with those of HDAC5 wild-type mice. Conclusions We reveal a novel role of HDAC5 in modulating the KLF2 transcriptional activation and eNOS expression. These findings suggest that HDAC5, a binding partner and modulator of KLF2, could be a new therapeutic target to prevent vascular endothelial dysfunction associated with cardiovascular diseases.

Atherosclerosis is a mechanobiology-related disease that preferentially develops in the aortic arch and arterial branches, which are exposed to disturbed/turbulent blood flow but less in thoracic aorta where the flow pattern is steady laminar flow (LF). Increasing evidence supports that steady LF with high shear stress is protective against atherosclerosis. However, the molecular mechanisms of LF-mediated atheroprotection remain incompletely understood. Hippo/YAP (yes-associated protein) pathway senses and effects mechanical cues and has been reported to be a master regulator of cell proliferation, differentiation, and tissue homeostasis. Here, we show that LF inhibits YAP activity in endothelial cells (ECs). We observed that YAP is highly expressed in mouse EC–enriched tissues (lung and aorta) and in human ECs. Furthermore, we found in apolipoprotein E deficient (ApoE -/- ) mice and human ECs, LF decreased the level of nuclear YAP protein and YAP target gene expression (connective tissue growth factor and cysteine-rich protein 61) through promoting Hippo kinases LATS1/2-dependent YAP (Serine 127) phosphorylation. Functionally, we revealed that YAP depletion in ECs phenocopying LF responses, reduced the expression of cell cycle gene cyclin A1 ( CCNA1 ) and proinflammatory gene CCL2 ( MCP-1 ). Taken together, we demonstrate that atheroprotective LF inhibits endothelial YAP activation, which may contribute to LF-mediated ECs quiescence and anti-inflammation.

Objective— Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion. Methods and Results— Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs. Conclusion— Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.

Rationale: The Rad-Gem/Kir-related family (RGKs) consists of small GTP-binding proteins that strongly inhibit the activity of voltage-gated calcium channels. Among RGKs, Rem1 is strongly and specifically expressed in cardiac tissue. However, the physiological role and regulation of RGKs, and Rem1 in particular, are largely unknown. Objective: To determine if Rem1 function is physiologically regulated by adrenergic signaling and thus impacts voltage-gated L-type calcium channel (VLCC) activity in the heart. Methods and Results: We found that activation of protein kinase D1, a protein kinase downstream of α 1 -adrenergic signaling, leads to direct phosphorylation of Rem1 at Ser18. This results in an increase of the channel activity and plasma membrane expression observed by using a combination of electrophysiology, live cell confocal microscopy, and immunohistochemistry in heterologous expression system and neonatal cardiomyocytes. In addition, we show that stimulation of α 1 -adrenergic receptor-protein kinase D1-Rem1 signaling increases transverse-tubule VLCC expression that results in increased L-type Ca 2+ current density in adult ventricular myocytes. Conclusion: The α 1 -adrenergic stimulation releases Rem1 inhibition of VLCCs through direct phosphorylation of Rem1 at Ser18 by protein kinase D1, resulting in an increase of the channel activity and transverse-tubule expression. Our results uncover a novel molecular regulatory mechanism of VLCC trafficking and function in the heart and provide the first demonstration of physiological regulation of RGK function.

Objective— Grb2-associated binder 1 (Gab1), a scaffolding adaptor protein, plays an important role in transmitting key signals that control cell growth, differentiation, and function from multiple tyrosine kinase receptors. The study was designed to investigate the role of endothelial Gab1 in angiogenesis and its underlying molecular mechanisms. Methods and Results— Using Cre-Lox recombination technology, we generated endothelial-specific Gab1 knockout (Gab1-ecKO) mice. Gab1-ecKO mice are viable and showed no obvious developmental defects in the vascular system. To analyze the role of Gab1 in postnatal angiogenesis, we used hindlimb ischemia and Matrigel plug models. We found that loss of endothelial Gab1 in mice dramatically impaired postnatal angiogenesis. Gab1-ecKO mice had impaired ischemia-initiated blood flow recovery, exhibited reduced angiogenesis, and were associated with marked limb necrosis. We further observed significant endothelial cell (EC) death in the ischemic hindlimb of Gab1-ecKO mice. Matrigel plug assay showed that hepatocyte growth factor (HGF)–mediated angiogenesis was inhibited in Gab1-ecKO mice. In vitro studies showed that Gab1 was required for HGF-induced EC migration, tube formation, and microvessel sprouting. Mechanistically, HGF stimulated Gab1 tyrosine phosphorylation in ECs, leading to activation of extracellular regulated MAP kinase 1/2 and Akt, which are angiogenic and survival signaling. Conclusion— Gab1 is essential for postnatal angiogenesis through mediating angiogenic and survival signaling.

Dynamic nucleocytoplasmic shuttling of class IIa histone deacetylases (HDACs) is a fundamental mechanism regulating gene transcription. Recent studies have identified several protein kinases that phosphorylate HDAC5, leading to its exportation from the nucleus. However, the negative regulatory mechanisms for HDAC5 nuclear exclusion remain largely unknown. Here we show that cAMP-activated protein kinase A (PKA) specifically phosphorylates HDAC5 and prevents its export from the nucleus, leading to suppression of gene transcription. PKA interacts directly with HDAC5 and phosphorylates HDAC5 at serine 280, an evolutionarily conserved site. Phosphorylation of HDAC5 by PKA interrupts the association of HDAC5 with protein chaperone 14-3-3 and hence inhibits stress signal-induced nuclear export of HDAC5. An HDAC5 mutant that mimics PKA-dependent phosphorylation localizes in the nucleus and acts as a dominant inhibitor for myocyte enhancer factor 2 transcriptional activity. Molecular manipulations of HDAC5 show that PKA-phosphorylated HDAC5 inhibits cardiac fetal gene expression and cardiomyocyte hypertrophy. Our findings identify HDAC5 as a substrate of PKA and reveal a cAMP/PKA-dependent pathway that controls HDAC5 nucleocytoplasmic shuttling and represses gene transcription. This pathway may represent a mechanism by which cAMP/PKA signaling modulates a wide range of biological functions and human diseases such as cardiomyopathy.

Fluid shear stress generated by steady laminar blood flow protects vessels from atherosclerosis. Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) are fluid shear stress–responsive genes and key mediators in flow anti-inflammatory and antiatherosclerotic actions. However, the molecular mechanisms underlying flow induction of KLF2 and eNOS remain largely unknown. Here, we show a novel role of histone deacetylase 5 (HDAC5) in flow-mediated KLF2 and eNOS expression. We found for the first time that fluid shear stress stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a calcium/calmodulin-dependent pathway. Consequently, flow induced the dissociation of HDAC5 and myocyte enhancer factor-2 (MEF2) and enhanced MEF2 transcriptional activity, which leads to expression of KLF2 and eNOS. Adenoviral overexpression of a HDAC5 phosphorylation–defective mutant (Ser259/Ser498 were replaced by Ala259/Ala498, HDAC5-S/A), which shows resistance to flow-induced nuclear export, suppressed flow-mediated MEF2 transcriptional activity and expression of KLF2 and eNOS. Importantly, HDAC5-S/A attenuated the flow-inhibitory effect on monocyte adhesion to endothelial cells. Taken together, our results reveal that phosphorylation-dependent derepression of HDAC5 mediates flow-induced KLF2 and eNOS expression as well as flow anti-inflammation, and suggest that HDAC5 could be a potential therapeutic target for the prevention of atherosclerosis.

Objective— Histone acetylation/deacetylation plays an important role in the control of gene expression, tissue growth, and development. In particular, histone deacetylases 7 (HDAC7), a member of class IIa HDACs, is crucial in maintaining vascular integrity. However, whether HDAC7 is involved in the processes of vascular endothelial signaling and angiogenesis remains unclear. Here, we investigated the role of HDAC7 in vascular endothelial growth factor (VEGF) signaling and angiogenesis. Methods and Results— We show for the first time that VEGF stimulated phosphorylation of HDAC7 at the sites of Ser178, Ser344, and Ser479 in a dose- and time-dependent manner, which leads to the cytoplasmic accumulation of HDAC7. Using pharmacological inhibitors, siRNA, and adenoviruses carrying dominant-negative mutants, we found that phospholipase Cγ/protein kinase C/protein kinase D1 (PKD1)-dependent signal pathway mediated HDAC7 phosphorylation and cytoplasmic accumulation by VEGF. Infection of ECs with adenoviruses encoding a mutant of HDAC7 specifically deficient in PKD1-dependent phosphorylation inhibited VEGF-induced angiogenic gene expression, including matrix metalloproteinases MT1-matrix metalloproteinase (MMP) and MMP10. Moreover, HDAC7 and its targeting genes were involved in VEGF-stimulated endothelial cell migration, tube formation, and microvessel sprouting. Conclusions— Our results demonstrate that VEGF stimulates PKD1-dependent HDAC7 phosphorylation and cytoplasmic accumulation in endothelial cells modulating gene expression and angiogenesis.

Background— Angiotensin II (Ang II) induces the phenotypic modulation and hypertrophy of vascular smooth muscle cells (VSMCs), which is implicated in the pathogenesis of hypertension, atherosclerosis, and diabetes. In this study, we tested the hypothesis that histone deacetylases 5 (HDAC5) and its signal pathway play a role in Ang II-induced VSMC hypertrophy. Methods and Results— VSMCs were isolated from the thoracic aortas of male Sprague-Dawley rats and treated with Ang II. We found that Ang II rapidly stimulated phosphorylation of HDAC5 at Serine259/498 residues in a time- and dose- dependent manner. Ang II receptor-1, protein kinase C, and protein kinase D1 (PKD1) mediated HDAC5 phosphorylation. Furthermore, we observed that Ang II stimulated HDAC5 nuclear export, which was dependent on its PKD1-dependent phosphorylation. Consequently, both inhibiting PKD1 and HDAC5 Serine259/498 to Alanine mutant significantly attenuated Ang II-induced myocyte enhancer factor-2 (MEF2) transcriptional activity and protein synthesis in VSMCs. Conclusion— These findings demonstrate for the first time that PKD1-dependent HDAC5 phosphorylation and nuclear export mediates Ang II-induced MEF2 activation and VSMC hypertrophy, and suggest that PKD1 and HDAC5 may emerge as potential targets for the treatment of pathological vascular hypertrophy.

Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PECAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PECAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PECAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK1/2, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PECAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction.

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stress=12 dyne/cm 2 ) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow.