Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3.

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-γ (IFN-γ) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-γ-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-γ also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.

Splicing of histone deacetylase 7 modulates smooth muscle cell proliferation and neointima formation through nuclear ß-catenin translocation.

OBJECTIVE: Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation and could have a role in SMC proliferation. In this study, we sought to examine the effect of 2 HDAC7 isoforms on SMC proliferation and neointima formation. METHODS AND RESULTS: We demonstrated that overexpression of unspliced HDAC7 (HDAC7u) could suppress SMC proliferation through downregulation of cyclin D1 and cell cycle arrest, whereas spliced HDAC7 (HDAC7s) could not. Small interfering RNA (siRNA)-mediated knockdown of HDAC7 increased SMC proliferation and induced nuclear translocation of ß-catenin. Additional experiments showed that only HDAC7u could bind to ß-catenin and retain it in the cytoplasm. Reporter gene assay and reverse transcription polymerase chain reaction revealed a reduction of ß-catenin activity in cells overexpressing HDAC7u but not HDAC7s. Deletion studies indicated that the C-terminal region of HDAC7u is responsible for the interaction with ß-catenin. However, the addition of amino acids to the N terminus of HDAC7u disrupted the binding, further strengthening our hypothesis that HDAC7s does not interact with ß-catenin. The growth factor platelet-derived growth factor-BB increased the splicing of HDAC7 while simultaneously decreasing the expression of HDAC7u. Importantly, in an animal model of femoral artery wire injury, we demonstrated that knockdown of HDAC7 by siRNA aggravates neointima formation in comparison with control siRNA. CONCLUSION: Our findings demonstrate that splicing of HDAC7 modulates SMC proliferation and neointima formation through ß-catenin nuclear translocation, which provides a potential therapeutic target in vascular disease.

Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow.

BACKGROUND: Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development. METHODS AND RESULTS: En face staining of aortas from apolipoprotein E-knockout mice revealed increased expression of HDAC3, specifically in these branching areas in vivo, whereas rapid upregulation of HDAC3 protein was observed in endothelial cells exposed to disturbed flow in vitro. Interestingly, phosphorylation of HDAC3 at serine/threonine was observed in these cells, suggesting that disturbed flow leads to posttranscriptional modification and stabilization of the HDAC3 protein. Coimmunoprecipitation experiments showed that HDAC3 and Akt form a complex. Using a series of constructs harboring deletions, we found residues 136 to 206 of HDAC3 to be crucial in this interaction. Enforced expression of HDAC3 resulted in increased phosphorylation of Akt and upregulation of its kinase activity. In line with these findings, knockdown of HDAC3 with lentiviral vectors (shHDAC3) led to a dramatic decrease in cell survival accompanied by apoptosis in endothelial cells. In aortic isografts of apolipoprotein E-knockout mice treated with shHDAC3, a robust atherosclerotic lesion was formed. Surprisingly, 3 of the 8 mice that received shHDAC3-infected grafts died within 2 days after the operation. Miller staining of the isografts revealed disruption of the basement membrane and rupture of the vessel. CONCLUSIONS: Our findings demonstrated that HDAC3 serves as an essential prosurvival molecule with a critical role in maintaining the endothelial integrity via Akt activation and that severe atherosclerosis and vessel rupture in isografted vessels of apolipoprotein E-knockout mice occur when HDAC3 is knocked down.

Protein kinase C{delta} deficiency accelerates neointimal lesions of mouse injured artery involving delayed reendothelialization and vasohibin-1 accumulation.

OBJECTIVE: To use protein kinase C (PKC) δ-knockout mice to investigate the role of PKCδ in lesion development and to understand the underlying mechanism of the vascular disease. METHODS AND RESULTS: PKCδ functions as a signal transducer mediating several essential functions of cell proliferation and apoptosis. However, the effect of PKCδ on neointimal formation in wire-injured vessels is unknown. Three weeks after wire injury of femoral arteries, neointimal lesions were significantly increased in PKCδ(-/-) mice compared with PKCδ(+/+) animals. Immunohistochemical staining revealed that total numbers of smooth muscle cells and macrophages in the lesions of PKCδ(-/-) mice were markedly elevated without changing the ratio of these 2 cell types. To further elucidate the mechanisms of PKCδ-mediated increase in the lesion, an in vivo endothelial migration model was established to evaluate endothelial wound healing after wire injury. Data showed that reendothelialization of the injured vessel was markedly delayed in PKCδ(-/-) mice; this coincided with more severe intimal hyperplasia. Migration of endothelial cells cultivated from cardiac tissue was markedly reduced in the absence of PKCδ, whereas no difference in proliferation or apoptosis was detected. Inhibition of PKCδ activity or protein expression by small hairpin RNA (shRNA) in cultured endothelial cells confirmed the defective migratory phenotype. Interestingly, vasohibin-1, an antiangiogenesis protein, was elevated in endothelial cells derived from PKCδ(-/-) mice, which was mainly because of delayed protein degradation mediated by PKCδ. Downregulation of vasohibin-1 restored the migration rate of PKCδ(-/-) endothelial cells to a similar level as PKCδ(+/+) cells. CONCLUSIONS: PKCδ deficiency enhances neointimal formation, which is associated with delayed reendothelialization and involves increased cellular vasohibin-1 accumulation.

HES1 is a novel interactor of the Fanconi anemia core complex.

Fanconi anemia (FA) proteins are thought to play a role in chromosome stability and repair of DNA cross-links; however, these functions may not fully explain the developmental abnormalities and bone marrow failure that are characteristic of FA individuals. Here we associate the FA proteins with the Notch1 developmental pathway through a direct protein-protein interaction between the FA core complex and the hairy enhancer of split 1 (HES1). HES1 interaction with FA core complex members is dependent on a functional FA pathway. Cells depleted of HES1 exhibit an FA-like phenotype that includes cellular hypersensitivity to mitomycin C (MMC) and lack of FANCD2 monoubiquitination and foci formation. HES1 is also required for proper nuclear localization or stability of some members of the core complex. Our results suggest that HES1 is a novel interacting protein of the FA core complex.

Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer.

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs of FancC(-/-) mice. Using this approach, we show that FancC(-/-) HSCs were efficiently corrected. Intrafemoral gene transfer of the FancC gene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

The fanconi anemia core complex acts as a transcriptional co-regulator in hairy enhancer of split 1 signaling.

Mutations in one of the 13 Fanconi anemia (FA) genes cause a progressive bone marrow failure disorder associated with developmental abnormalities and a predisposition to cancer. Although FA has been defined as a DNA repair disease based on the hypersensitivity of patient cells to DNA cross-linking agents, FA patients develop various developmental defects such as skeletal abnormalities, microphthalmia, and endocrine abnormalities that may be linked to transcriptional defects. Recently, we reported that the FA core complex interacts with the transcriptional repressor Hairy Enhancer of Split 1 (HES1) suggesting that the core complex plays a role in transcription. Here we show that the FA core complex contributes to transcriptional regulation of HES1-responsive genes, including HES1 and the cyclin-dependent kinase inhibitor p21(cip1/waf1). Chromatin immunoprecipitation studies show that the FA core complex interacts with the HES1 promoter but not the p21(cip1/waf1) promoter. Furthermore, we show that the FA core complex interferes with HES1 binding to the co-repressor transducin-like-Enhancer of Split, suggesting that the core complex affects transcription both directly and indirectly. Taken together these data suggest a novel function of the FA core complex in transcriptional regulation.

Lack of self-renewal capacity in Fancc-/- stem cells after ex vivo expansion.

Treatments of the hematological manifestation in Fanconi anemia (FA) are first supported by attempts to stimulate hematopoiesis with androgens or hematopoietic growth factors. However, the long-term curative treatment of the hematological manifestation in FA patients is bone marrow (BM) or cord blood stem cell transplantation. The success rate for BM transplantation is fairly high with HLA-matched sibling donors but is, unfortunately, low with HLA-matched unrelated donors. An alternative curative treatment for those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Because FA patients have reduced numbers of stem/progenitor cells, ex vivo expansion of hematopoietic stem cells would be a crucial step in gene transfer protocols. Using the FA mouse model, Fancc-/-, we tested the ability of CD34- hematopoietic stem cells to support ex vivo expansion. We determined that Fancc-/- CD34- stem cells have reduced reconstitution ability and markedly reduced self-renewal ability after culture, as shown by secondary transplants. These results indicate that FA stem cells may not be well suited for ex vivo expansion before gene transfer or transplantation protocols.