Yeast-derived particulate beta-glucan induced angiogenesis via regulating PI3K/Src and ERK1/2 signaling pathway.

The importance of ß-glucan from S. cerevisiae in angiogenesis has not been well studied. We investigated whether ß-glucan induces angiogenesis through PI3K/Src and ERK1/2 signaling pathway in HUVECs. We identified that ß-glucan induced phosphorylation of PI3K, Src, Akt, eNOS, and ERK1/2. Subsequently, we found that this phosphorylation increased the viability of HUVECs. We also observed that stimulation of ß-glucan promoted the activity of MEF2 and MEF2-dependent pro-angiogenic genes, including EGR2, EGR3, KLF2, and KLF4. Additionally, the role of ß-glucan in angiogenesis was confirmed using in vitro and ex vivo experiments including cell migration, capillary-like tube formation and mouse aorta ring assays. To determine the effect of ß-glucan on the PI3K/Akt/eNOS and ERK1/2 signaling pathway, PI3K inhibitor wortmannin and ERK1/2 inhibitor SCH772984 were used. Through the Matrigel plug assay, we confirmed that ß-glucan significantly increased angiogenesis in vivo. Taken together, our study demonstrates that ß-glucan promotes angiogenesis via through PI3K and ERK1/2 signaling pathway.

HDAC5-mediated exosomal Maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma.

BACKGROUND: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. METHODS: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. RESULTS: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. CONCLUSIONS: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma.

Angiogenic adipokine C1q-TNF-related protein 9 ameliorates myocardial infarction via histone deacetylase 7-mediated MEF2 activation.

C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine and has high potential as a therapeutic target. However, the role of CTRP9 in cardiovascular disease pathogenesis remains unclear. We found CTRP9 to induce HDAC7 and p38 MAPK phosphorylation via tight regulation of AMPK in vascular endothelial cells, leading to angiogenesis through increased MEF2 activity. The expression of CTRP9 and atheroprotective MEF2 was decreased in plaque tissue of atherosclerotic patients and the ventricle of post-infarction mice. CTRP9 treatment inhibited the formation of atherosclerotic plaques in ApoE KO and CTRP9 KO mice. In addition, CTRP9 induced significant ischemic injury prevention in the post-MI mice. Clinically, serum CTRP9 levels were reduced in patients with MI compared with healthy controls. In summary, CTRP9 induces a vasoprotective response via the AMPK/HDAC7/p38 MAPK pathway in vascular endothelial cells, whereas its absence can contribute to atherosclerosis and MI. Hence, CTRP9 may represent a valuable therapeutic target and biomarker in cardiovascular diseases.

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression.

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.

miR-3189-targeted GLUT3 repression by HDAC2 knockdown inhibits glioblastoma tumorigenesis through regulating glucose metabolism and proliferation.

BACKGROUND: Epigenetic regulations frequently appear in Glioblastoma (GBM) and are highly associated with metabolic alterations. Especially, Histone deacetylases (HDACs) correlates with the regulation of tumorigenesis and cell metabolism in GBM progression, and HDAC inhibitors report to have therapeutic efficacy in GBM and other neurological diseases; however, GBM prevention and therapy by HDAC inhibition lacks a mechanism in the focus of metabolic reprogramming. METHODS: HDAC2 highly express in GBM and is analyzed in TCGA/GEPIA databases. Therefore, HDAC2 knockdown affects GBM cell death. Analysis of RNA sequencing and qRT-PCR reveals that miR-3189 increases and GLUT3 decreases by HDAC2 knockdown. GBM tumorigenesis also examines by using in vivo orthotopic xenograft tumor models. The metabolism change in HDAC2 knockdown GBM cells measures by glucose uptake, lactate production, and OCR/ECAR analysis, indicating that HDAC2 knockdown induces GBM cell death by inhibiting GLUT3. RESULTS: Notably, GLUT3 was suppressed by increasing miR-3189, demonstrating that miR-3189-mediated GLUT3 inhibition shows an anti-tumorigenic effect and cell death by regulating glucose metabolism in HDAC2 knockdown GBM. CONCLUSIONS: Our findings will demonstrate the central role of HDAC2 in GBM tumorigenesis through the reprogramming of glucose metabolism by controlling miR-3189-inhibited GLUT3 expression, providing a potential new therapeutic strategy for GBM treatment.

Mannoproteins from Saccharomyces cerevisiae stimulate angiogenesis by promoting the akt-eNOS signaling pathway in endothelial cells.

Mannoproteins (MPs) are a major component of yeast cell walls and consist of high levels of mannose in covalent complexes with proteins. MPs complexly enhance the immune system. We previously isolated a mutant yeast, K48L3, with a higher yield of MP from its cell wall than wild-type Saccharomyces cerevisiae, YPH499. We determined that K48L3 induces the release of nitric oxide in macrophage cells. The present study reports nitric-oxide-mediated angiogenesis by MP from K48L3 and the induction of the Akt/eNOS signal pathway. Western blotting and RT-PCR were used to demonstrate that MP treatment resulted in the upregulation of p-Akt, p-eNOS, and angiogenesis-mediated gene expression. Moreover, the angiogenesis activity of the MPs was demonstrated using three angiogenesis assays, namely, a cell migration assay, a tube-forming assay, and an ex vivo aorta ring assay. Thus, this study demonstrates for the first time that MPs from S. cerevisiae K48L3 induce angiogenesis in HUVECs via the Akt-eNOS-dependent signaling pathway.

Radiation-Induced Phosphorylation of Serine 360 of SMC1 in Human Peripheral Blood Mononuclear Cells.

In the event of a mass casualty radiation scenario, biodosimetry has the potential to quantify individual exposures for triaging and providing dose-appropriate medical intervention. Structural maintenance of chromosomes 1 (SMC1) is phosphorylated in response to ionizing radiation. The goal of this study was to develop a new biodosimetry method using SMC1 phosphorylation as a measure of exposure to radiation. In the initial experiments, two normal human cell lines (WI-38VA-13 and HaCaT) and four lymphoblastoid cell lines were irradiated, and the levels of SMC1 phosphorylation at Ser-360 and Ser-957 were assessed using Western blotting. Subsequently, similar experiments were performed using peripheral blood mononuclear cells (PBMCs) obtained from 20 healthy adults. Phosphorylation of SMC1 at Ser-957 and Ser-360 was increased by exposure in a dose-dependent manner, peaked at 1-3 h postirradiation and then decreased gradually. Ser-360 was identified as a new phosphorylation site and was more sensitive to radiation than Ser-957, especially at doses below 1 Gy. Our results demonstrate a robust ex vivo response of phospho-SMC1-(Ser-360) to ionizing radiation in human PBMCs. Detection of phosphorylation at Ser-360 in SMC1 could be used as a marker of radiation exposure. Our findings suggest that it is feasible to measure blood cell-based changes in the phosphorylation level of a protein as an ex vivo radiation exposure detection method, even after low-dose exposure.

Programmed cell death 5 suppresses AKT-mediated cytoprotection of endothelium.

Programmed cell death 5 (PDCD5) has been associated with human cancers as a regulator of cell death; however, the role of PDCD5 in the endothelium has not been revealed. Thus, we investigated whether PDCD5 regulates protein kinase B (PKB/AKT)-endothelial nitric oxide synthase (eNOS)-dependent signal transduction in the endothelium and affects atherosclerosis. Endothelial-specific PDCD5 knockout mice showed significantly reduced vascular remodeling compared with wild-type (WT) mice after partial carotid ligation. WT PDCD5 competitively inhibited interaction between histone deacetylase 3 (HDAC3) and AKT, but PDCD5L6R, an HDAC3-binding-deficient mutant, did not. Knockdown of PDCD5 accelerated HDAC3-AKT interaction, AKT and eNOS phosphorylation, and nitric oxide (NO) production in human umbilical vein endothelial cells. Moreover, we found that serum PDCD5 levels reflect endothelial NO production and are correlated with diabetes mellitus, high-density lipoprotein cholesterol, and coronary calcium in human samples obtained from the cardiovascular high-risk cohort. Therefore, we conclude that PDCD5 is associated with endothelial dysfunction and may be a novel therapeutic target in atherosclerosis.

Superior Efficacy and Selectivity of Novel Small-Molecule Kinase Inhibitors of T790M-Mutant EGFR in Preclinical Models of Lung Cancer.

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200-11. ©2017 AACR.

Paradoxical induction of growth arrest and apoptosis by EGF via the up-regulation of PTEN by activating Redox factor-1/Egr-1 in human lung cancer cells.

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

Shikonin Induces Apoptotic Cell Death via Regulation of p53 and Nrf2 in AGS Human Stomach Carcinoma Cells.

Shikonin, which derives from Lithospermum erythrorhizon, has been traditionally used against a variety of diseases, including cancer, in Eastern Asia. Here we determined that shikonin inhibits proliferation of gastric cancer cells by inducing apoptosis. Shikonin's biological activity was validated by observing cell viability, caspase 3 activity, reactive oxygen species (ROS) generation, and apoptotic marker expressions in AGS stomach cancer cells. The concentration range of shikonin was 35-250 nM with the incubation time of 6 h. Protein levels of Nrf2 and p53 were evaluated by western blotting and confirmed by real-time PCR. Our results revealed that shikonin induced the generation of ROS as well as caspase 3-dependent apoptosis. c-Jun-N-terminal kinases (JNK) activity was significantly elevated in shikonin-treated cells, thereby linking JNK to apoptosis. Furthermore, our results revealed that shikonin induced p53 expression but repressed Nrf2 expression. Moreover, our results suggested that there may be a co-regulation between p53 and Nrf2, in which transfection with siNrf2 induced the p53 expression. We demonstrated for the first time that shikonin activated cell apoptosis in AGS cells via caspase 3- and JNK-dependent pathways, as well as through the p53-Nrf2 mediated signal pathway. Our study validates in partly the contribution of shikonin as a new therapeutic approaches/ agent for cancer chemotherapy.

PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling.

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.

Chemopreventive Properties of Genipin on AGS Cell Line via Induction of JNK/Nrf2/ARE Signaling Pathway.

Roles of dietary phytochemicals in cancer chemoprevention via induction of nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated antioxidant enzymes have been well established in a number of studies. In this study, FACS analysis was used to reveal that the intracellular reactive oxygen species level decreased at 0-25 µM of genipin treatment. Furthermore, immunofluorescence analysis and Western blotting were used to demonstrate that genipin treatment resulted in the upregulation and nuclear translocation of Nrf2, as well as upregulation of gastrointestinal glutathione peroxidase. Finally, we found that C-Jun-NH2-kinase (JNK) was also dose-dependently activated, where depleting JNK by using a biochemical inhibitor indicated that JNK was upstream of Nrf2. Interestingly, the antioxidant effects were limited to the treatment in the lower dosage of genipin, where higher dosage of genipin treatment resulted in the increased reactive oxygen species level and cytotoxicity. Thus, this study demonstrates for the first time that lower dosage of genipin results in the induction of JNK/Nrf2/ARE signaling pathway and protection from cell death.

Induction of apoptosis by genipin inhibits cell proliferation in AGS human gastric cancer cells via Egr1/p21 signaling pathway.

Natural compounds are becoming important candidates in cancer therapy due to their cytotoxic effects on cancer cells by inducing various types of programmed cell deaths. In this study, we investigated whether genipin induces programmed cell deaths and mediates in Egr1/p21 signaling pathways in gastric cancer cells. Effects of genipin in AGS cancer cell lines were observed via evaluation of cell viability, ROS generation, cell cycle arrest, and protein and RNA levels of p21, Egr1, as well as apoptotic marker genes. The cell viability of AGS cells reduced by genipin treatment via induction of the caspase 3-dependent apoptosis. Cell cycle arrest was observed at the G2/M phase along with induction of p21 and p21-dependent cyclins. As an upstream mediator of p21, the transcription factor early growth response-1 (Egr1) upregulated p21 through nuclear translocation and binding to the p21 promoter site. Silencing Egr1 expression inhibited the expression of p21 and downstream molecules involved in apoptosis. We demonstrated that genipin treatment in AGS human gastric cancer cell line induces apoptosis via p53-independent Egr1/p21 signaling pathway in a dose-dependent manner.

Novel mechanism of gene transfection by low-energy shock wave.

Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

Endothelial Grb2-associated binder 1 is crucial for postnatal angiogenesis.

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. CONCLUSIONS: Gab1 is essential for postnatal angiogenesis through mediating angiogenic and survival signaling.

PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy.

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 stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS.

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.

Protein kinase D1, a new molecular player in VEGF signaling and angiogenesis.

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal and pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are incompletely understood. The protein kinase D1 (PKD1), a newly described calcium/calmodulin-dependent serine/threonine kinase, has been implicated in cell migration, proliferation and membrane trafficking. Increasing evidence suggests critical roles for PKD1-mediated signaling pathways in endothelial cells, particularly in the regulation of VEGF-induced angiogenesis. Recent studies show that class IIa histone deacetylases (HDACs) are PKD1 substrates and VEGF signal-responsive repressors of myocyte enhancer factor-2 (MEF2) transcriptional activation in endothelial cells. This review provides a guide to PKD1 signaling pathways and the direct downstream targets of PKD1 in VEGF signaling, and suggests important functions of PKD1 in angiogenesis.

VEGF stimulates HDAC7 phosphorylation and cytoplasmic accumulation modulating matrix metalloproteinase expression and angiogenesis.

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 Cgamma/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.