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DNA methyltransferase 3a (DNMT3a) is an important part of the epigenetic machinery that stabilizes patterns of activated T cell responses. We hypothesized that donor T cell DNMT3a regulates alloreactivity after allogeneic blood and marrow transplantation (allo-BMT). T cell conditional Dnmt3a KO mice were used as donors in allo-BMT models. Mice receiving allo-BMT from KO donors developed severe acute graft-versus-host disease (aGVHD), with increases in inflammatory cytokine levels and organ histopathology scores. KO T cells migrated and proliferated in secondary lymphoid organs earlier and demonstrated an advantage in trafficking to the small intestine. Donor T cell subsets were purified after BMT for whole-genome bisulfite sequencing (WGBS) and RNA-Seq. KO T cells had global methylation similar to that of WT cells, with distinct, localized areas of hypomethylation. Using a highly sensitive computational method, we produced a comprehensive profile of the altered epigenome landscape. Hypomethylation corresponded with changes in gene expression in several pathways of T cell signaling and differentiation. Additionally, Dnmt3a-KO T cells resulted in superior graft-versus-tumor activity. Our findings demonstrate a critical role for DNMT3a in regulating T cell alloreactivity and reveal pathways that control T cell tolerance. These results also provide a platform for deciphering clinical data that associate donor DNMT3a mutations with increased GVHD, decreased relapse, and improved survival.

CD4+ T cells lacking the mTORC1 activator Rheb fail to secrete IFN-γ under Th1 polarizing conditions. We hypothesized that this phenotype is due to defects in regulation of the canonical Th1 transcription factor T-bet at the level of protein phosphorylation downstream of mTORC1. To test this hypothesis, we employed targeted mass-spectrometry proteomic analysis–multiple reaction monitoring mass spectrometry. We used this method to detect and quantify predicted phosphopeptides derived from T-bet. By analyzing activated murine wild-type and Rheb-deficient CD4+ T cells, as well as murine CD4+ T cells activated in the presence of rapamycin, a pharmacologic inhibitor of mTORC1, we were able to identify six T-bet phosphorylation sites. Five of these are novel, and four sites are consistently dephosphorylated in both Rheb-deficient CD4+ T cells and T cells treated with rapamycin, suggesting mTORC1 signaling controls their phosphorylation. Alanine mutagenesis of each of the six phosphorylation sites was tested for the ability to impair IFN-γ expression. Single phosphorylation site mutants still support induction of IFN-γ expression; however, simultaneous mutation of three of the mTORC1-dependent sites results in significantly reduced IFN-γ expression. The reduced activity of the triple mutant T-bet is associated with its failure to recruit chromatin remodeling complexes to the Ifng gene promoter. These results establish a novel mechanism by which mTORC1 regulates Th1 differentiation, through control of T-bet phosphorylation.

DNMT3a is a de novo DNA methyltransferase expressed robustly after T-cell activation that regulates plasticity of CD4(+) T-cell cytokine expression. Here we show that DNMT3a is critical for directing early CD8(+) T-cell effector and memory fate decisions. Whereas effector function of DNMT3a knockout T cells is normal, they develop more memory precursor and fewer terminal effector cells in a T-cell intrinsic manner compared with wild-type animals. Rather than increasing plasticity of differentiated effector CD8(+) T cells, loss of DNMT3a biases differentiation of early effector cells into memory precursor cells. This is attributed in part to ineffective repression of Tcf1 expression in knockout T cells, as DNMT3a localizes to the Tcf7 promoter and catalyzes its de novo methylation in early effector WT CD8(+) T cells. These data identify DNMT3a as a crucial regulator of CD8(+) early effector cell differentiation and effector versus memory fate decisions.

Background Pulmonary dysfunction remains one of the greatest challenges to the optimization of care following allogeneic blood and marrow transplantation (alloBMT). Almost half of the pneumonias that occur early after alloBMT are noninfectious, termed idiopathic pneumonia syndrome (IPS). Injury/activation to vascular endothelial cells (ECs) are believed to be directly related to several post-BMT complications, including IPS. Hence, identifying inflammatory mechanisms contributing to EC damage and strategies to protect EC integrity and function have significant merit. We have demonstrated that TNFα contributes directly to EC injury and regulates the chemokine milieu and influx of donor cells into the lung. Laboratory insights led to clinical trials of TNFα inhibition for IPS. Many, but not all, patients with IPS respond to TNFα inhibition, underscoring the need for continued research. Results Analysis of the plasma proteome from samples derived from 3 clinical studies revealed that compared to unaffected BMT controls, patients with IPS showed marked elevations in the expression of signature cytokines, including TNFα and MCP-1. Elevated expression of molecules associated with EC injury, angiopoietin (Ang)-2, VCAM-1, and E-selectin, was also noted. Ang-2 has been shown to destabilize and further sensitize ECs to the effects of TNFα, causing enhanced expression of E- and P-selectin. Serum Ang-2 levels were elevated during IPS and correlated with response to anti-TNFα therapy. In an acute lung injury (ALI) model, IV injection of LPS to naive B6 mice results in enhanced mRNA expression of TNFα, IL-6, Ang-2, E-, and P-selectin in whole lung homogenates, and the expression of Ang-2 is regulated in part by TNFα and IL-6. EC activation was associated with increased BALF total protein and cellularity. Similar findings were noted in alloBMT mice with IPS. We hypothesized that strategies to maintain EC integrity would reduce the severity of ALI and IPS. Defibrotide (DF) is FDA approved for the treatment of BMT patients with VOD and evidence of renal or pulmonary injury. DF is believed to stabilize activated ECs and protect them from further injury. The administration of DF (50 mg/kg) IV before and after LPS injection significantly reduced mRNA expression of TNFα, IL6, Ang-2, E-, and P-selectin compared to controls. BALF showed decreased cellularity, reflecting less EC damage and leak. AlloBMT mice were treated from day -1 through day 14 with DF ip (at a dose comparable to IV) either QD or BID, and lungs were harvested from D18 to 21. Compared to controls, DF treatment reduced mRNA expression of TNFα, IL6, Ang-2, E-, and P- selectin, BALF cellularity and protein content, and lung pathology score. Conclusion The administration of DF modulates EC injury in models of ALI and IPS. Combining cytokine inhibition with agents that stabilize EC integrity may represent a novel therapeutic strategy for patients with IPS.

Osteoporosis and osteonecrosis are associated with corticosteroid treatment, but the pathophysiologies are unclear. We hypothesized that mature adipocytes present within the bone marrow compartment play a key role in the development of both diseases. Adipocytes have recognized regulatory effects on bone viability and healing by releasing signaling molecules called adipokines. Our purpose was to evaluate whether dexamethasone alters adipokine expression in differentiated bone-marrow-derived adipocytes. Adipocytes differentiated from mouse D1 mesenchymal stromal cells were treated with dexamethasone (10(-5), 10(-6), 10(-7), or 10(-8)M) or with diluent alone (controls) for up to 6days. Using real-time polymerase chain reaction and enzyme-linked immunosorbent assay analyses, six key adipokines and the transcription factor HIF-1α were evaluated. Dexamethasone treatment increased PAI-1 protein expression with increased mRNA expression at 4days, while decreasing HIF-1α mRNA expression and protein concentrations. VEGF A mRNA expression was increased at 4days for most dexamethasone concentrations, with minimal changes in protein levels. Dexamethasone increased adiponectin mRNA expression and protein levels at 4 and 6days and decreased leptin, interleukin-6, and tumor necrosis factor α mRNA expression at all time periods. Dexamethasone treatment of bone-marrow-derived adipocytes resulted in detectable changes in mRNA expression and protein levels of adipokines and HIF-1α. The detected adipokine alterations could be important early events in the pathogenesis of steroid-induced osteonecrosis and osteoporosis.

The increased bone marrow lipid deposition in steroid-associated bone loss diseases indicates that abnormalities in fat metabolism are associated with disease development. Recent studies have suggested that bone marrow adipocytes are secretory cells and that they may release substances that have an inhibitory effect on the differentiation and function of osteoblasts. We hypothesized that exposure of bone-marrow-derived adipocytes to corticosteroids exacerbates their deleterious effects on osteoblast metabolism and function. Adipocytes and osteoblasts derived from a human mesenchymal stem cell line (240L) were co-cultured in the absence of direct cell contact with or without dexamethasone treatment. After 6days of co-culture, osteoblasts demonstrated significantly lower levels of function based on lower mineralization, alkaline phosphatase activity and expression of osteogenic (Runx2, osteocalcin) mRNA marker. Dexamethasone treatment resulted in significantly lower levels of osteoblastic function compared with co-cultured cells without dexamethasone. Furthermore, conditioned media from dexamethasone-treated adipocytes induced a similar toxic effect and increased apoptosis involving activation of caspases 3/7 compared with conditioned media without dexamethasone treatment. Within the conditioned media, a substantial increase in the levels of leptin and two saturated fatty acids (FAs; stearate and palmitate) was observed after dexamethasone treatment. Although leptin supplementation failed to induce the inhibitory effect on osteoblasts, similar toxic results were produced with stearate and palmitate treatment, and an increase in intracellular reactive oxygen species was observed. Stearate- and palmitate-induced apoptosis was blocked by a reactive oxygen species scavenger pyrrolidine dithiocarbamate. These data show that saturated FAs secreted from adipocytes induce lipotoxic effects via mechanisms that may involve reactive oxygen species accumulation in osteoblasts. Our results suggest that inhibition of saturated FA secretion would protect osteoblasts against adipocytes in corticosteroid-associated bone loss diseases.

Aims It has been well demonstrated that phosphodiesterase-5A (PDE5A) is expressed in smooth muscle cells and plays an important role in regulation of vascular tone. The role of endothelial PDE5A, however, has not been yet characterized. The present study was undertaken to determine the presence, localization, and potential physiologic significance of PDE5A within vascular endothelial cells. Methods and results We demonstrate primary location of human, mouse, and bovine endothelial PDE5A at or near caveolae. We found that the spatial localization of PDE5A at the level of caveolin-rich lipid rafts allows for a feedback loop between endothelial PDE5A and nitric oxide synthase (NOS3). Treatment of human endothelium with PDE5A inhibitors resulted in a significant increase in NOS3 activity, whereas overexpression of PDE5A using an adenoviral vector, both in vivo and in cell culture, resulted in decreased NOS3 activity and endothelium-dependent vasodilation. The molecular mechanism responsible for these interactions is primarily regulated by cGMP-dependent second messenger. PDE5A overexpression also resulted in a significant decrease in protein kinase 1 (PKG1) activity. Overexpression of PKG1 rapidly activated NOS3, whereas silencing of the PKG1 gene with siRNA inhibited both NOS3 phosphorylation (S1179) and activity, indicating a novel role for PKG1 in direct regulation of NOS3. Conclusion Our data collectively suggest another target for PDE5A inhibition in endothelial dysfunction and provide another physiologic significance for PDE5A in the modulation of endothelial-dependent flow-mediated vasodilation. Using both in vitro and in vivo models, as well as human data, we show that inhibition of endothelial PDE5A improves endothelial function.

Osteonecrosis is a disease that is characterized by lesions of dead bone, most frequently detected in the subchondral bone of the convex side of the major diarthrodial joints (hips, knees, shoulders, and talus). High-dose corticosteroid therapy is one major risk factor for osteonecrosis. The pathological appearance of osteonecrosis of the femoral head, however, is fairly consistent regardless of the etiology. Depending on the stage of the disease, this includes evidence of bone marrow edema, lipocyte and bone cell necrosis, fibrosis, creeping substitution (new bone lying on dead bone), and the presence of osteoporosis [1]. Hemorrhage within the bone marrow compartment has also been observed in patient specimens from corticosteroid-associated osteonecrosis [2]. Previous investigators have suggested that corticosteroid-associated osteonecrosis has more rapid progression than other etiologies [1, 3]. The mechanisms involved in the development of osteonecrosis [ON] are difficult to explore from clinical samples as they provide a single snapshot at one time point subsequent to the onset of the disease, usually months after the onset of the disease when the symptoms first appear. While some investigators have used corticosteroid-associated animal models to evaluate different potential treatments [4–6], it is not clear whether the results can be extrapolated to the human condition. A better understanding of the pathophysiological mechanisms is needed.

Activation of the vascular endothelium occurs in many clinical scenarios such as inflammatory or infectious conditions (sepsis), reperfusion injury, and transplant graft rejection.1–5 Under such circumstances, endothelial activation is primarily induced by cytokines such as tumor necrosis factor (TNF) and interleukin-1β (IL-1β) and vascular permeability factors such as vascular endothelial growth factor (VEGF) that upregulate a number of genes including prothrombotic factors, chemokines, and cell adhesion molecules, many of which are dependent on the action of the pleiotropic transcription factor nuclear factor-κB (NF-κB).2,6–11 Activated endothelium is compromised in its structural and functional integrity, leading to transmigration of leukocytes into the vessel wall, plasma leakage, and thrombosis. Angiopoeitin-1 (Ang1) is a vasculogenic factor that induces endothelial cell sprouting, migration, and network formation,12–15 coordinated processes that are crucial in the development of new blood vessels. Angiopoietins signal via the Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) family of receptor tyrosine kinases, of which Tie2 is endothelial-cell specific. In addition to its important role in vasculogenesis, Ang1, through Tie2, suppresses activation of endothelial cells as evidenced by its inhibition of vascular cell adhesion molecules and procoagulant tissue factor expression.16,17 These effects confer on Ang1 potent antiinflammatory properties.18–20 Although Ang1 affects the expression of many NF-κB–responsive genes, a functional and mechanistic link between Ang1 and NF-κB signaling has not been previously shown. The report by Hughes et al21 in this issue of …

In vitro modeling of adipose tissue is essential for the study of adipogenesis and related diseases as well as for the development of surgical reconstruction procedures and tissue-engineering applications. Peroxisome proliferator activated receptor ? (PPAR?) has been shown to play an integral role in stimulating adipogenesis. There are several established ligands for PPAR?, including rosiglitazone. D1 cells, a multipotential cell line derived from mouse bone marrow, were treated with increasing (0.1, 1, 10, and 30 µM) concentrations of rosiglitazone in DMEM for 48 hours followed by treatment by DMEM alone for up to 15 days. All doses of rosiglitazone stimulated the accumulation of lipids ,which was notable by day 6. The adipogenic effect of rosiglitazone was maximized at doses of 10 and 30 µM. Adipogenesis for rosiglitazone-treated cells was greater than that for cells treated with dexamethasone, a conventional method used to stimulate adipogenesis. Significantly higher levels of triglyceride-G (TG) and mature adipocyte markers (PPAR-, adipocyte fatty acid-binding protein) were observed with rosiglitazone treatment after 6 days. Cytokines in the supernatants were analyzed by multiplex-based ELISA assays at day 6 after treatment; these cells release adiponectin, resistin, PAI-1, MCP-1, and VEGF with either rosiglitazone or dexamethasone treatment. However, rosiglitazone treatment had lower osteocalcin release than did the control. This study provides evidence that rosiglitazone treatment is a reliable method that can be used to induce adipogenesis of D1 cells, a pluripotential cell line from mouse bone marrow.

Introduction: cGMP specific phosphodiesterase type 5A (PDE5A) has a key role in limiting the effect of the second messenger cGMP as demonstrated by the use of the specific inhibitor, sildenafil. However, the mechanism by which PDE5 activity is regulated is not fully understood. Recently, it has been reported that PDE5A specifically degrades the pool of cGMP produced by the nitric oxide (NO) stimulated soluble guanylate cyclase. NO is known to regulate other proteins in this pathway through S-nitrosylation, a redox modification where an NO group is covalently added to the sulfur atom of a cysteine. Here we test the hypothesis that PDE5 activity is regulated by S-nitrosylation and determine the critical modified cysteine residues. Methods: Rates of cGMP hydrolysis were measured by an in vitro activity assay using purified mouse flag-PDE5 treated with the NO-donor, S-Nitrosoglutathione (GSNO, 1–100μM) or control. The S-nitrosylation status of PDE5A was determined by biotin switch assay of cell lysates over expressing flag-PDE5A using the equivalent conditions. After NO modifications were replaced with biotin moieties, labeled proteins or trypsin digested peptides were captured with streptavidin agarose and evaluated by western blot or tandem mass spectrometry, respectively. Results: PDE5A activity was found to be decreased in a dose dependent manner with NO donor inducing a 25.0 ± 3.8% (n=4, p Conclusions: These data indicates that PDE5A activity is negatively regulated by S-nitrosylaion at C181 which may contribute to the overall regulation of cGMP signaling in the cell. This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).

Background— The B vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an antioxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and postreperfusion injury. Methods and Results— Wistar rats were pretreated with either FA (10 mg/d) or placebo for 1 week and then underwent in vivo transient left coronary artery occlusion for 30 minutes with or without 90 minutes of reperfusion (total n=131; subgroups used for various analyses). FA (4.5×10 −6 mol/L IC) pretreatment and global ischemia/reperfusion (30 minutes/30 minutes) also were performed in vitro (n=28). After 30 minutes of ischemia, global function declined more in controls than in FA-pretreated rats (ΔdP/dtmax, −878±586 versus −1956±351 mm Hg/s placebo; P =0.03), and regional thickening was better preserved (37.3±5.3% versus 5.1±0.6% placebo; P =0.004). Anterior wall perfusion fell similarly (−78.4±9.3% versus −71.2±13.8% placebo at 30 minutes), yet myocardial high-energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA pretreatment (ATP: control, 2740±58 nmol/g; ischemia, 947±55 nmol/g; ischemia plus FA, 1332±101 nmol/g; P =0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA pretreatment but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124±280 cpm/mg FA versus 5898±474 cpm/mg placebo; P =0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8±1.2% versus 60.3±4.1% placebo area at risk; P Conclusions— FA pretreatment blunts myocardial dysfunction during ischemia and ameliorates postreperfusion injury. This is coupled to preservation of high-energy phosphates, reducing subsequent reactive oxygen species generation, eNOS-uncoupling, and postreperfusion cell death.

The p66shc adaptor protein mediates age-associated oxidative stress. We examined the role of p66shc in endothelial nitric oxide synthase (eNOS) signaling. Overexpression of p66shc inhibited eNOS-dependent NO production. RNAi-mediated down-regulation of endogenous p66shc led to activation of the proto-oncogene ras, and Akt kinase, with a corresponding increase in phosphorylation of eNOS at S1177 (S1179 on bovine eNOS). In rat aortic rings, down-regulation of p66shc suppressed the vasoconstrictor response to phenyephrine that was abrogated by treatment with the NOS inhibitor l-NAME, and enhanced vasodilation induced by sub-maximal doses of acetylcholine. These findings highlight a pivotal role for p66shc in inhibiting endothelial NO production, and endothelium-dependent vasorelaxation, that may provide important mechanistic information about endothelial dysfunction seen with aging.

The dual-function protein apurinic/apyrmidinic endonuclease/redox factor-1 (APE1/ref-1) is essential for DNA repair and also governs the reductive activation of many redox-sensitive transcription factors. We examined the role of APE1/ref-1 in regulation of endothelium-dependent tone and systemic blood pressure. APE1/ref-1 +/− mice have impaired endothelium-dependent vasorelaxation, reduced vascular NO levels, and are hypertensive. APE1/ref-1 upregulates H-ras expression and leads to H-ras-mediated, phosphoinositide-3 kinase/Akt kinase-dependent calcium sensitization of endothelial NO synthase (eNOS), stimulating NO production. The reducing property of APE1/ref-1 is essential for upregulation of H-ras and for the calcium sensitization of eNOS. These findings uncover a novel physiological role for APE1/ref-1 in regulating vascular tone by governance of eNOS activity and bioavailable NO.

A variety of mutations in the gene encoding the GHRH receptor (GHRHR) that are predicted to alter protein structure or function have been recently described in patients with isolated GH deficiency type IB. In the present report we describe a patient with isolated GH deficiency type IB who was heterozygous for two novel mutations in this gene: a missense mutation in codon 329 that replaces lysine with glutamic acid (K329E) and an A→C transversion (position −124) in one of the two sites of the promoter region that binds the pituitary-specific transcription factor Pit-1, which is required for GHRHR expression. Chinese hamster ovary cells that were transfected with a cDNA encoding the K329E GHRHR expressed the receptor but failed to show a cAMP response after treatment with GHRH, confirming the lack of functionality. To test the effect of the A→C mutation at position −124 of the promoter, we transfected rat GH3 pituitary cells, which express endogenous Pit-1, with plasmids in which the luciferase reporter gene was under the control of either the wild-type or the mutant promoter. GH3 cells expressing the mutant promoter showed significantly less luciferase activity than cells expressing the wild-type promoter. DNA-binding studies confirmed that the A→C base change markedly reduces DNA binding to the Pit-1 protein. These results demonstrate that mutations in the GHRHR are not limited to the coding sequence and that promoter mutations that impair Pit-1 binding can reduce expression of the GHRHR gene.