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Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood neutrophilia, marrow granulocyte hyperplasia, hepatosplenomegaly, and driver mutations in the colony-stimulating factor 3 receptor (CSF3R). Designation of activating CSF3R mutations as a defining genomic abnormality for CNL has led to increased recognition of the disease. However, the natural history of CNL remains poorly understood with most patients reported being of older age, lacking germline data, and having poor survival, in part due to transformation to acute leukemia. CSF3R driver mutations in most patients with CNL have been reported to be acquired, although rare cases of germline mutations have been described. Here, we report the largest pedigree to date with familial CNL, spanning four generations with affected family members ranging in age from 4 to 53 years, none of whom have transformed to acute leukemia. A heterozygous T618I CSF3R mutation was identified in peripheral blood and mesenchymal stromal cells from the proband and in all affected living family members, while the unaffected family members tested were homozygous wild type. We show that the T618I mutation also confers a survival advantage to neutrophils in an MCL1-dependent manner. Collectively, these data provide additional insights into the natural history of familial CNL arising from T618I CSF3R mutations and suggest that enhanced neutrophil survival also contributes to the high neutrophil count observed in patients with CNL., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

The revised International Prognostic Index (R-IPI) is an important prognostic tool in diffuse large B cell lymphoma (DLBCL); however, outcomes can vary markedly within R-IPI groups, and additional prognostic markers are needed. We conducted a prospective observational study to evaluate the circulating immature myeloid (IM) cell subsets and cytokine profiles of 31 patients with newly diagnosed DLBCL before and after chemoimmunotherapy. Among circulating IM cells, myeloid-derived suppressor cells (MDSCs) were the predominant cell type (73.8% ± 26%). At baseline, circulating monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) were predominantly mutually exclusive. Patients with DLBCL clustered into three distinct immunotypes according to MDSC levels and subtype predominance: M-MDSC high , PMN-MDSC high , and MDSC low . The M-MDSC high immunotype was associated with the germinal center B cell-like (GCB) subtype and elevated serum IL-8 and MIP-1α levels. PMN-MDSC high was associated with the non-GCB subtype and elevated IL-8, MCP-1, IP-10, TNFα, and IL-1Ra levels. Standard chemoimmunotherapy partially reduced M-MDSC distribution across the MDSC low and M-MDSC high groups. By contrast, among the MDSC low and PMN-MDSC high groups, PMN-MDSCs persisted after treatment. Two high-risk patients with non-GCB DLBCL and MDSC low immunotype experienced early disease recurrence within 12 months of treatment completion. This study demonstrates that distinct types of MDSCs are associated with subtypes of DLBCL. MDSC levels are dynamic and may be associated with disease status. Persistence of PMN-MDSCs among high-risk patients with DLBCL may be associated with early relapse., Competing Interests: Conflict of Interest Disclosure DMF has received research funding from TeneoBio and Celgene and honoraria from Cytognos. BH is an advisory board member for BeiGene, ADC Therapeutics, and Morphosys. TM has received consulting fees from Kite Pharma. RJ has received consulting fees from AbbVie, AstraZeneca, BeiGene, Genentech, Janssen, Loxo/Lilly, Morphosys, Pharmacyclics, and TG Therapeutics; has served on the speaker's bureau for AbbVie, Adaptive Biotech, AstraZeneca, BeiGene, Janssen, Loxo/Lilly, Pharmacyclics, and TG Therapeutics; and has received research funding from TG Therapeutics, AstraZeneca, Loxo/Lilly, and AbbVie. NG reports serving as a consultant or advisor for Seattle Genetics, TG Therapeutics, AstraZeneca, Pharmacyclics/Janssen, Bristol-Myers Squibb, Gilead Sciences, BeiGene, Incyte, Karyopharm, Roche/Genentech, Novartis, Loxo/Lilly, Genmab, Adaptive Biotechnologies, Syncopation, and Lava Therapeutics; participation on speakers bureau for Kite/Gilead, AstraZeneca, Bristol-Myers Squibb, Pharmacyclics/Janssen, and Epizyme; research funding from Pharmacyclics, TG Therapeutics, Genentech/Roche, Bristol-Myers Squibb/Celgene, Gilead Sciences, Morphosys, and AbbVie. The other authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2023 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

CYP2C19-guided voriconazole dosing reduces pharmacokinetic variability, but many patients remain subtherapeutic. The aim of this study was to evaluate the effect of candidate genes and a novel CYP2C haplotype on voriconazole trough concentrations in patients receiving CYP2C19-guided dosing. This is a retrospective candidate gene study in allogeneic hematopoietic cell transplant (HCT) patients receiving CYP2C19-guided voriconazole dosing. Patients were genotyped for ABCB1, ABCG2, CYP2C9, CYP3A4, CYP3A5, and the CYP2C haplotype. Of 185 patients, 36% were subtherapeutic (of which 79% were normal or intermediate metabolizers). In all patients, CYP2C19 (p < 0.001), age (p = 0.018), and letermovir use (p = 0.001) were associated with voriconazole concentrations. In the subset receiving 200 mg daily (non-RM/UMs), CYP2C19 (p = 0.004) and ABCG2 (p = 0.015) were associated with voriconazole concentrations; CYP2C19 (p = 0.028) and letermovir use (p = 0.001) were associated with subtherapeutic status. CYP2C19 phenotype and letermovir use were significantly associated with subtherapeutic voriconazole concentrations and may be used to improve voriconazole precision dosing, while further research is needed to clarify the role of ABCG2 in voriconazole dosing., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Treatment of patients with multiple myeloma (MM) in first relapse remains a challenge. This phase II study combined elotuzumab (Elo) with carfilzomib, lenalidomide, and dexamethasone (KRd) for treatment of MM in first relapse with the aim of improving efficacy., Methods: Enrolled patients received Elo-KRd induction for 4 cycles, and Elo-lenalidomide maintenance until progression. The primary endpoint was VGPR or better (≥VGPR) postinduction. Secondary endpoints were MRD by flow cytometry, OS, PFS, and safety. Correlatives included characterization of the impact of Elo-KRd on NK and T cell subsets via flow cytometry. Target accrual of 40 patients was not met due to COVID-19 pandemic., Results: Of 15 patients enrolled, 10 (67%) had high-risk features (del17p, t[4;14], t[14;16], 1q gain/amplification, plasma cell leukemia, extramedullary MM, or functional high risk), 12 (80%) were lenalidomide-refractory, and 5 (33.3%) bortezomib-refractory. Postinduction ≥VGPR was 7/15 (46.7%) and MRD-negative (10 -5 ) rate 20%. Overall response during study was 80%, including ≥VGPR as best response of 53.3%. At median follow-up of 28.2 (range, 3.8 to 44.2) months, the median PFS was 11.5 months (95% CI 1.9, 18), and median OS not reached (95% CI 10.1, NA). No new safety concerns were reported. Elo-KRd treatment did not augment NK cell distribution or activity in blood or bone marrow. Effector CD4+ and CD8+ T cells significantly decreased postinduction, with concomitant acquisition of T central memory phenotype, particularly at a high rate in ≥VGPR group., Conclusion: A short course of Elo-KRd induction followed by Elo-lenalidomide maintenance demonstrated activity in predominantly lenalidomide-refractory and / or high-risk MM. The results with this well-tolerated combination are comparable to other contemporary approved triplet combinations., Competing Interests: Disclosure Bhutani: Research Funding: Janssen R&D, MedImmune, Legend Biotech, Amgen, Celularity, Adaptive Biotechnologies, Bristol Myers Squibb / Celgene, Bluebird bio, Millenium, Cerecor, Takeda; Consultancy: Sanofi, Janssen, Atrash: Honoraria and Research Funding: GSK; Honoraria and Speakers Bureau: Celgene, Sanofi, Janssen; Honoraria: Takeda; Membership on an entity's Board of Directors or advisory committees: Janssen; Research Funding: Amgen. Paul: Membership on an entity's Board of Directors or advisory committees: Abbvie, Janssen, Regeneron; Speakers Bureau: Amgen, Symanowski: Consultancy: Camarus, Eli Lilly and Co, CARsgen, Immatics, Voorhees: Consultancy and Honoraria: Abbvie, Amgen, BMS, GSK, Karyopharm, Novartis, Oncopeptides, Pfizer, Sanofi, SecuraBio, Usmani: Research Funding: AbbVie, Amgen, BMS, Celgene, EdoPharma, Genentech, Gilead, GSK, Janssen Pharmaceuticals, Oncopeptides, Sanofi, Seagen Inc, formerly Seattle Genetics, Inc, Secura Bio, Inc, SkylineDX, Takeda, TeneoBio, Amgen, Array Biopharma, BMS, Celgene, GSK, Janssen; Consultancy: Amgen, Array Biopharma, BMS, Celgene, GSK, Janssen Pharmaceuticals, Merck, Pharmacyclics, Sanofi, Seagen Inc, formerly Seattle Genetics, Inc, SkylineDX, Takeda; Speakers Bureau: Amgen, BMS, Janssen Pharmaceuticals, Sanofi. *Other authors declare no COI., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Administration of plerixafor with granulocyte-colony stimulating factor (G-CSF) mobilizes CD34+ cells much more effectively than G-CSF alone, but cost generally limits plerixafor use to patients at high risk of insufficient CD34+ cell collection based on low peripheral blood (PB) CD34+ counts following 4 days of G-CSF. We analyzed costs associated with administering plerixafor to patients with higher day 4 CD34+ cell counts to decrease apheresis days and explored the use of a fixed split dose of plerixafor instead of weight-based dosing. We analyzed 235 patients with plasma cell disorders or non-Hodgkin's lymphoma who underwent progenitor cell mobilization and autologous hematopoietic cell transplantation (AHCT) between March 2014 and December 2017. Two hundred ten (89%) received G-CSF plus Plerixafor and 25 (11%) received G-CSF alone. Overall, 180 patients (77%) collected in 1 day, 53 (22%) in 2 days and 2 (1%) in 3 days. Based on our data, we present a probabilistic algorithm to identify patients likely to require more than one day of collection using G-CSF alone. CD34+ cell yield, ANC and platelet recovery were not significantly different between fixed and standard dose plerixafor. Plerixafor enabled collection in 1 day and with estimated savings of $5000, compared to patients who did not receive plerixafor and required collection for three days. While collection and processing costs and patient populations vary among institutions, our results suggest re-evaluation of current algorithms., (© 2021 Wiley Periodicals LLC.)

Colony-stimulating factor 3 receptor (CSF3R) encodes the receptor for granulocyte colony-stimulating factor (G-CSF), a cytokine vital for granulocyte proliferation and differentiation. Acquired activating heterozygous variants in CSF3R are the main cause of chronic neutrophilic leukemia, a hyperproliferative disorder. In contrast, biallelic germ line hypomorphic variants in CSF3R are a rare cause of severe congenital neutropenia, a hypoproliferative condition. The impact of heterozygous germ line CSF3R variants, however, is unknown. We identified CSF3R as a new germ line hematologic malignancy predisposition gene through analysis of 832 next-generation sequencing tests conducted in 632 patients with hematologic malignancies. Among germ line CSF3R variants, 3 were abnormal in functional testing, indicating their deleterious nature. p.Trp547* was identified in 2 unrelated men with myelodysplastic syndromes diagnosed at 76 and 33 years of age, respectively. p.Trp547* is a loss-of-function nonsense variant in the extracellular domain that results in decreased CSF3R messenger RNA expression and abrogation of CSF3R surface expression and proliferative responses to G-CSF. p.Ala119Thr is a missense variant found in 2 patients with multiple myeloma and acute lymphoblastic leukemia, respectively. This variant is located between the extracellular immunoglobulin-like and cytokine receptor homology domains and results in decreased G-CSF sensitivity. p.Pro784Thr was identified in a 67-year-old man with multiple myeloma. p.Pro784Thr is a missense variant in the cytoplasmic domain that inhibits CSF3R internalization, producing a gain-of-function phenotype and G-CSF hypersensitivity. Our findings identify germ line heterozygous CSF3R variants as risk factors for development of myeloid and lymphoid malignancies., (© 2020 by The American Society of Hematology.)

Activating mutations in the membrane-proximal region of the colony-stimulating factor 3 receptor (CSF3R) are a hallmark of chronic neutrophilic leukemia (CNL) with the T618I mutation being most common. The mechanisms underlying constitutive activation of the T618I CSF3R and its signal propagation are poorly understood. Ligand-independent activation of the T618I CSF3R has previously been attributed to loss of receptor O-glycosylation and increased receptor dimerization. Here, we show that the T618I CSF3R is indeed glycosylated but undergoes enhanced spontaneous internalization and degradation that results in a marked decrease in its surface expression. Inhibition of the proteasome dramatically increases expression of the O-glycosylated T618I CSF3R. We also demonstrate that the O-glycosylated wild-type CSF3R is tyrosine phosphorylated in response to ligand but constitutively phosphorylated in cells expressing T618I CSF3R. Constitutive tyrosine phosphorylation of the O-glycosylated T618I receptor form correlated with activation of JAK2 and both the mutant receptor and JAK2 were found to be constitutively ubiquitinated. These observations provide novel insights into the mechanisms of oncogenic signaling by T618I CSF3R mutations in CNL., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Three annotated CSF3R mRNA splice variants have been described. CSF3R-V1 is the wild-type receptor, while CSF3R-V4 is a truncated form increased in some patients with AML. CSF3R-V3 mRNA was identified in placenta more than 20 years ago, but remains largely uncharacterized due to the lack of a suitable detection assay. Using a novel digital PCR method to quantitate expression of each CSF3R mRNA splice variant in hematopoietic cells, CSF3R-V1 was most highly expressed followed by CSF3R-V3. Functional assays revealed expression of V3 alone conferred a hypoproliferative phenotype associated with defective JAK-STAT activation. However, coexpression of V1 with V3 rescued proliferative responses. Comparative analysis of V3/V1 expression in CD34+ cells from healthy donors and patients with AML revealed a statistically significant increase in the V3/V1 ratio only in the subset of patients with AML harboring SRSF2 mutations. Knockout of SRFS2 in KG-1 and normal CD34+ cells decreased the V3/V1 ratio. Collectively, these data are the first to demonstrate expression of the CSF3R-V3 splice variant in primary human myeloid cells and a role for SRSF2 in modulating CSF3R splicing. Our findings provide confirmatory evidence that CSF3R is a target of SRSF2 mutations, which has implications for novel treatment strategies for SRSF2-mutated myeloid malignancies.

Despite improvements in cancer early detection and treatment, metastatic breast cancer remains deadly. Current therapeutic approaches have very limited efficacy in patients with triple negative breast cancer. Among the many mechanisms associated that contribute to cancer progression, signaling through the CXCL12-CXCR4 is an essential step in cancer cell migration. We previously demonstrated the formation of CXCL12-CXCL4 heterodimers (Carlson et al., 2013). Here, we investigated whether CXCL12-CXCL4 heterodimers alter tumor cell migration. CXCL12 alone dose-dependently promoted the MDA-MB 231 cell migration (p < .05), which could be prevented by blocking the CXCR4 receptor. The addition of CXCL4 inhibited the CXCL12-induced cell migration (p < .05). Using NMR spectroscopy, we identified the CXCL4-CXCL12 binding interface. Moreover, we generated a CXCL4-derived peptide homolog of the binding interface that mimicked the activity of native CXCL4 protein. These results confirm the formation of CXCL12-CXCL4 heterodimers and their inhibitory effects on the migration of breast tumors cells. These findings suggest that specific peptides mimicking heterodimerization of CXCL12 might prevent breast cancer cell migration., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pharmacogenetics influences oral tacrolimus exposure; however, little data exist regarding i.v. tacrolimus. We investigated the impact of genetic polymorphisms in CYP3A4, CYP3A5, and ABCB1 on i.v. tacrolimus exposure and toxicity in adult patients receiving an allogeneic hematopoietic stem cell transplant for hematologic malignancies. Germline DNA was extracted from buccal swabs and genotyped for CYP3A4, CYP3A5, and ABCB1 polymorphisms. Continuous i.v. infusion of tacrolimus .03 mg/kg/day was initiated on day +5 post-transplant, and steady-state blood concentrations were measured 4days later. We evaluated the association between phenotypes and prevalence of nontherapeutic target concentrations (below or above 5 to 15 ng/mL) as well as tacrolimus-related toxicities. Of 63 patients, 28.6% achieved the target concentration; 71.4% were >15ng/mL, which was more common in CYP3A4 intermediate/normal metabolizers (compared with rapid) and those with at least 1 ABCB1 C2677T loss-of-function allele (P < .05). ABCB1 C2677T was significantly associated with concentrations >15ng/mL (odds ratio, 6.2; 95% confidence interval, 1.8 to 23.6; P = .004) and tacrolimus-related toxicities (odds ratio, 7.5; 95% confidence interval, 1.6 to 55.2; P = .02). ABCB1 C2677T and CYP3A4 are important determinants of i.v. tacrolimus exposure, whereas ABCB1 C2677T also impacts tacrolimus-related toxicities in stem cell transplants., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Data indicate reversal of immune dysfunction with active treatment; however, the precise contribution of specific immune effector and immune suppressor components to achieve a minimal residual disease (MRD) state and immunomodulatory drug-mediated immunomodulatory effects in multiple myeloma (MM) patients remains poorly understood. In this prospective proof-of-principle study we sought to determine the dynamic alterations in natural killer (NK), NK-T, and T cells, including maturation and activating/inhibitory repertoire associated with MRD pos versus MRD neg status after autologous stem cell transplantation (ASCT) and during lenalidomide-based maintenance therapy. Of the 46MM patients enrolled, 36 had bone marrow MRD assessment 60+ days post-ASCT, 30 had longitudinal blood immunotyping during maintenance (pretherapy and after cycles 1, 3, and 6), and 20 had both MRD assessment and longitudinal immunotyping. Multicolor flow cytometry was used for MRD and immunotyping. Although the absolute number of NK cells was significantly lower in patients with MRD pos response, phenotypically NK cells in these patients displayed higher expression of activating receptors KIRDS4 and decreased expression of inhibitory molecules NKG2A compared with the MRD neg group. Furthermore, we observed significantly lower frequencies of T cells displaying KIR3DL1 in MRD pos versus MRD neg patients. Longitudinal immunotyping during lenalidomide maintenance showed loss of mature NK effector function, augmentation of NK-T effector function, and acquisition of PD1 independent anergic state. Our findings also suggest skewing of T cells toward an exhausted state during the maintenance phase in MRD pos patients. Put together, these observations provide a distinctive signature for MRD neg and MRD pos groups. These data support exploration of immune profiling in prospective clinical trials according to MRD-defined responses to identify patients that may benefit from maintenance intensification/modification or maintenance withdrawal., (Copyright © 2018 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Immunomodulatory derivatives (IMiDs), along with proteasome inhibitors, are key components of treatment regimens for multiple myeloma. Nonetheless, outcomes vary among treated individuals. Drug-specific gene-expression profile (GEP) signatures that aid the prediction of favourable and unfavourable outcomes can provide patients with the most effective therapy for their individual disease. We aimed to develop and validate a gene expression signature to suggest which patients would benefit most from IMiD-based therapies., Methods: For this exploratory retrospective study, we selected a cohort of patients with newly diagnosed or relapsed or refractory multiple myeloma who were treated in clinical trials with IMiD-containing regimens. Cohorts were eligible if they had publicly available GEP data from patients' bone marrow plasma cells, with long-term follow-up and clinicopathological data. In the development stage of the model, we identified 176 IMiD response genes that were differentially expressed before and after IMiD exposure using pharmacogenomic GEP data from patients who had bone marrow samples taken before and 48 h after a test dose exposure with thalidomide (n=42), lenalidomide (n=18), or pomalidomide (n=18). 14 of these genes had p values less than 0·05 for associations with progression-free survival in patients who received thalidomide in induction and maintenance therapy in the Total Therapy (TT) 2 trial (ie, the training cohort). We combined the 14 genes to create a continuous IMiD-14 score and an optimal cutoff. The subgroup with an IMiD-14 score higher than the cutoff was deemed to be IMiD-resistant. We obtained validation cohorts from four studies of IMiD combination regimens: the TT3a trial (thalidomide in induction and maintenance), the TT3b trial (thalidomide in induction and lenalidomide in maintenance), the TT6 trial (thalidomide in induction and lenalidomide in maintenance), and the vincristine, doxorubicin, and dexamethasone (VAD) group of the HOVON65/GMMG-HD4 trial (thalidomide in maintenance). The primary endpoint was to show the prognostic value of the IMiD-14 gene signature for progression-free survival., Findings: In the training cohort, progression-free survival was significantly shorter in the 83 patients with IMiD-14 high scores than in the 92 patients with IMiD-14 low scores; 3 year progression-free survival was 52% (95% CI 42-64) for the IMiD-14 high group versus 85% (78-92) for the IMiD-14 low group, with a hazard ratio (HR) of 2·51 (95% CI 1·72-3·66; p<0·0001). These findings were supported by the results in the validation cohorts, TT3a (115 patients with IMiD-14 high vs 160 patients with IMiD-14 low; 3 year progression-free survival 63% [95% CI 55-73] vs 87% [82-92]; HR 1·54 [1·11-2·15], p=0·010), TT3b (77 patients with IMiD-14 high vs 89 patients with IMiD-14 low; 62% [52-74] vs 80% [72-89]; HR 2·07 [1·28-3·34], p=0·0024), TT6 (20 patients with IMiD-14 high vs 36 patients with IMiD-14 low; 39% [22-68] vs 74% [61-90]; HR 2·40 [1·09-5·30], p=0·026), and the VAD group of HOVON65/GMMG-HD4 (65 patients with IMiD-14 high vs 77 patients with IMiD-14 low; 16% [9-28] vs 54% [44-67]; HR 2·29 [1·52-3·45], p<0·0001)., Interpretation: Our results suggest that the IMiD-14 model has prognostic value in patients with multiple myeloma who are treated with IMiDs. Some genes in the model could provide novel targets for IMiD resistance and therapeutic intervention. The IMiD-14 model warrants evaluation in prospective studies., Funding: Conquer Cancer Foundation ASCO Young Investigator Award and the Carolinas Myeloma Research Fund., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1., Competing Interests: The authors have declared that no competing interests exist.

Most patients with acute myeloid leukemia can be induced into complete remission, but postremission treatment is required for cure. The choice of postremission therapy in a majority of nonelderly patients, between intensive chemotherapy and allogeneic hematopoietic cell transplantation, is largely determined by the results of conventional cytogenetic analysis. In 45% of patients with a normal karyotype, the presence or absence of specific molecular mutations should be used to determine the prognosis and postremission treatment. In addition, the identification of mutations may indicate a role for targeted intervention, including following transplantation., (Copyright © 2015 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.)

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

Background and Purpose: Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC)., Experimental Approach: BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured., Key Results: Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO., Conclusion and Implications: Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases., (© 2014 The British Pharmacological Society.)

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an O(2)-dependent manner. This regulates NO levels in the vascular wall; however, the underlying molecular basis of this O(2)-dependent NO consumption remains unclear. While cytoglobin (Cygb) was discovered a decade ago, its physiological function remains uncertain. Cygb is expressed in the vascular wall and can consume NO in an O(2)-dependent manner. Therefore, we characterize the process of the O(2)-dependent consumption of NO by Cygb in the presence of the cellular reductants and reducing systems ascorbate (Asc) and cytochrome P(450) reductase (CPR), measure rate constants of Cygb reduction by Asc and CPR, and propose a reaction mechanism and derive a related kinetic model for this O(2)-dependent NO consumption involving Cygb(Fe(3+)) as the main intermediate reduced back to ferrous Cygb by cellular reductants. This kinetic model expresses the relationship between the rate of O(2)-dependent consumption of NO by Cygb and rate constants of the molecular reactions involved. The predicted rate of O(2)-dependent consumption of NO by Cygb is consistent with experimental results supporting the validity of the kinetic model. Simulations based on this kinetic model suggest that the high efficiency of Cygb in regulating the NO consumption rate is due to the rapid reduction of Cygb by cellular reductants, which greatly increases the rate of consumption of NO at higher O(2) concentrations, and binding of NO to Cygb, which reduces the rate of consumption of NO at lower O(2) concentrations. Thus, the coexistence of Cygb with efficient reductants in tissues allows Cygb to function as an O(2)-dependent regulator of NO decay.

Heat-shock factor 1 (HSF-1), a transcription factor for heat-shock proteins (HSPs), is known to interfere with the transcriptional activity of many oncogenic factors. In the present work, we have discovered that HSF-1 ablation induced the multidrug resistance gene, MDR1b, in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug-resistant cancer cells. The increase in P-gp enhanced the extrusion of doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Dox-induced left ventricular (LV) dysfunction was significantly reduced in HSF-1(-/-) mice. DNA-binding activity of NF-κB was higher in HSF-1(-/-) mice. IκB, the NF-κB inhibitor, was depleted due to enhanced IκB kinase (IKK)-α activity. In parallel, MDR1b gene expression and a large increase in P-gp and lowering Dox loading were observed in HSF-1(-/-) mouse hearts. Moreover, application of the P-gp antagonist, verapamil, increased Dox loading in HSF-1(-/-) cardiomyocytes, deteriorated cardiac function in HSF-1(-/-) mice, and decreased survival. MDR1 promoter activity was higher in HSF-1(-/-) cardiomyocytes, whereas a mutant MDR1 promoter with heat-shock element (HSE) mutation showed increased activity only in HSF-1(+/+) cardiomyocytes. However, deletion of HSE and NF-κB binding sites diminished luminescence in both HSF-1(+/+) and HSF-1(-/-) cardiomyocytes, suggesting that HSF-1 inhibits MDR1 activity in the heart. Thus, because high levels of HSF-1 are attributed to poor prognosis of cancer, systemic down-regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapy-induced cardiotoxicity and enhance cancer prognosis.

Extracellular heat shock proteins (eHsps) in the circulation have recently been found to activate both apoptotic and protective signaling in the heart. However, the role of eHsps in doxorubicin (Dox)-induced heart failure has not yet been studied. The objective of the present study was to determine how Dox affects circulating eHsp25 in blood plasma and how eHsp25 affects Dox-induced dilated cardiomyopathy. Wild-type mice [HSF-1(+/+)] were pretreated with 100 μl of heterozygous heat shock factor-1 [HSF-1(+/-)] mouse plasma (which contained 4-fold higher eHsp25 compared with wild-type mice), HSF-1(+/+) plasma, or saline, before treatment with Dox (6 mg/kg). After 4 weeks of this treatment protocol, HSF-1(+/-) plasma-pretreated mice showed increased eHsp25 in plasma and improved cardiac function (percentage of fractional shortening 37.3 ± 2.1 versus 26.4 ± 4.0) and better life span (31 ± 2 versus 22 ± 3 days) compared with the HSF-1(+/+) plasma or saline-pretreated mice. Preincubation of isolated adult cardiomyocytes with HSF-1(+/-) plasma or recombinant human Hsp27 (rhHsp27) significantly reduced Dox-induced activation of nuclear factor-κB and cytokine release and delayed cardiomyocyte death. Moreover, when cardiomyocytes were incubated with fluorescence-tagged rhHsp27, a saturation in binding was observed, suggesting that eHsp25 can bind to surface receptors. Competitive assays with a Toll-like receptor 2 (TLR2) antibody reduced the rhHSP27 binding, indicating that Hsp25 interacts with TLR2. In conclusion, transfusion of Hsp25-enriched blood plasma protected the heart from Dox-induced cardiotoxicity. Hsp25 antagonized Dox binding to the TLR2 receptor on cardiomyocytes.

Tetrahydrobiopterin (BH(4)) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH(4) is also an anti-oxidant being a good "scavenger" of oxidative species. NADPH oxidase, xanthine oxidase, and mitochondrial enzymes producing reactive oxygen species (ROS) can induce elevated oxidant stress and cause BH(4) oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH(4) degradation, a sensitive method for monitoring pteridine redox-state changes is required. Considering that the conventional fluorescence method is an indirect method requiring conversion of all pteridines to oxidized forms, it would be beneficial to use a rapid quantitative assay for the individual detection of BH(4) and its related pteridine metabolites. To study, in detail, the BH(4) oxidative pathways, a rapid direct sensitive HPLC assay of BH(4) and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH(4) autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH(4) transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH(2)), predominates with oxygen-induced BH(4) autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH(2)), are largely produced during hydroxyl radical-driven BH(4) oxidation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A(2B) adenosine receptor antagonist, MRS 1754, but not the A(1) selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A(2B) receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3β that prevents detrimental opening of the mitochondrial permeability transition pore., (2011 Elsevier Ltd. All rights reserved.)

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (BH(4)), NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH(4) oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH(4) depletion alone. In the current study, we investigated the effects of both BH(4) depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH(4) does not result in eNOS oxidase activity, whereas BH(4) oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms, which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches, we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using small interfering RNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH(4) oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH(4) depletion increased eNOS-derived superoxide to 165% of that observed with BH(4) oxidation. This study identifies a critical role of caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease-associated changes in caveolin-1 expression may contribute to endothelial dysfunction.

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(•-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(•-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O(2)(•-) generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O(2)(•-) generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mM) or hyperglycemia (30 mM). With hyperglycemia treatment, O(2) consumption rate was reduced (from V(O(2)max) = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10(6) cells), whereas in heat-shocked cells, O(2) consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10(6) cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.

Treating cancer patients with chemotherapeutics, such as doxorubicin (Dox), cause dilated cardiomyopathy and congestive heart failure because of oxidative stress. On the other hand, heat shock factor-1 (HSF-1), a transcription factor for heat shock proteins (Hsps), is also known to be activated in response to oxidative stress. However, the possible role of HSF-1 activation and the resultant Hsp25 in chemotherapeutic-induced heart failure has not been investigated. Using HSF-1 wild-type (HSF-1(+/+)) and knock-out (HSF-1(-/-)) mice, we tested the hypothesis that activation of HSF-1 plays a role in the development of Dox-induced heart failure. Higher levels of Hsp25 and its phosphorylated forms were found in the failing hearts of Dox-treated HSF-1(+/+) mice. More than twofold increase in Hsp25 mRNA level was found in Dox-treated hearts. Proteomic analysis showed that there is accumulation and aggregation of Hsp25 in Dox-treated failing hearts. Additionally, Hsp25 was found to coimmunoprecipitate with p53 and vice versa. Further studies indicated that the Dox-induced higher levels of Hsp25 transactivated p53 leading to higher levels of the pro-apoptotic protein Bax, but other p53-related proteins remained unaltered. Moreover, HSF-1(-/-) mice showed significantly reduced Dox-induced heart failure and higher survival rate, and there was no change in Bax upon treating with Dox in HSF-1(-/-) mice. From these results we propose a novel mechanism for Dox-induced heart failure: increased expression of Hsp25 because of oxidant-induced activation of HSF-1 transactivates p53 to increase Bax levels, which leads to heart failure.

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO(-)) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH(4)); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO(-)-induced eNOS dysfunction and their dose dependence. Exposure of human eNOS to ONOO(-) resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH(4) were oxidized during exposure to ONOO(-); however, full oxidation of protein-bound biopterin required higher ONOO(-) levels. Additionally, ONOO(-) triggered changes in the UV/visible spectrum and heme content of the enzyme. Preincubation of eNOS with BH(4) decreased dimer destabilization and heme alteration. Addition of BH(4) to the ONOO(-)-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO(-) treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH(4) does not require zinc occupancy of the zinc-thiolate cluster. While ONOO(-) treatment was observed to induce loss of Zn binding, this cannot account for the loss of enzyme activity. Therefore, ONOO(-)-induced eNOS inactivation is primarily due to oxidation of BH(4) and irreversible destruction of the heme/heme center.

Inducible NOS (iNOS) is induced in diseases associated with inflammation and oxidative stress, and questions remain regarding its regulation. We demonstrate that reactive oxygen/nitrogen species (ROS/RNS) dose-dependently regulate iNOS function. Tetrahydrobiopterin (BH4)-replete iNOS was exposed to increasing concentrations of ROS/RNS and activity was measured with and without subsequent BH4 addition. Peroxynitrite (ONOO(-)) produced the greatest change in NO generation rate, approximately 95% decrease, and BH4 only partially restored this loss of activity. Superoxide (O2(.-)) greatly decreased NO generation, however, BH4 addition restored this activity. Hydroxyl radical ((.)OH) mildly decreases NO generation in a BH4-dependent manner. iNOS was resistant to H2O2 with only slightly decreased NO generation with up to millimolar concentrations. In contrast to the inhibition of NO generation, ROS enhanced O2(.-) production from iNOS, while ONOO(-) had the opposite effect. Thus, ROS promote reversible iNOS uncoupling, while ONOO(-) induces irreversible enzyme inactivation and decreases both NO and O2(.-) production., (2009 Elsevier Inc. All rights reserved.)

Background: The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in maintaining homeostatic levels of circulating neutrophils (PMN). The mechanisms modulating G-CSFR surface expression to prevent chronic neutrophilia are poorly understood. Here, we report that neutrophil elastase (NE) proteolytically cleaves the G-CSFR on human PMN and blocks G-CSFR-mediated granulopoiesis in vitro., Methods: Human peripheral blood PMN isolated from healthy donors were incubated with NE. Expression of the G-CSFR was analyzed by flow cytometry and western blot analyses. Detection of G-CSFR cleavage products from the culture supernatants was also performed. Human bone marrow mononuclear cells were also cultured in the presence or absence of NE to determine its effects on the proliferation of granulocyte-macrophage colony forming units (CFU-GM)., Results: Treatment of PMN with NE induced a time-dependent decrease in G-CSFR expression that correlated with its degradation and the appearance of proteolytic cleavage fragments in conditioned media. Immunoblot analysis confirmed the G-CSFR was cleaved at its amino-terminus. Treatment of progenitor cells with NE prior to culture inhibited the growth of granulocyte-macrophage colony forming units., Conclusions: These findings indicate that in addition to transcriptional controls and ligand-induced internalization, direct proteolytic cleavage of the G-CSFR by NE also downregulates G-CSFR expression and inhibits G-CSFR-mediated granulopoiesis in vitro. Our results suggest that NE negatively regulates granulopoiesis through a novel negative feedback loop.

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis, but the mechanisms controlling its surface expression are poorly understood. Recent studies using transfected cell lines have suggested the activated G-CSFR is routed to the lysosome and not the proteasome. Here, we examined the role of the ubiquitin/proteasome system in regulating G-CSFR surface expression in both ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme and in primary human neutrophils. We show that the G-CSFR is constitutively ubiquitinated, which increases following ligand binding. In the absence of a functional E1 enzyme, ligand-induced internalization of the receptor is inhibited. Pre-treatment of ts20 transfectants with either chloroquine or MG132 inhibited ligand-induced G-CSFR degradation, suggesting a role for both lysosomes and proteasomes in regulating G-CSFR surface expression in this cell line. In neutrophils, inhibition of the proteasome but not the lysosome was found to inhibit internalization/degradation of the activated G-CSFR. Collectively, these data demonstrate the requirement for a functional ubiquitin/proteasome system in G-CSFR internalization and degradation. Our results suggest a prominent role for the proteasome in physiologic modulation of the G-CSFR, and provide further evidence for the importance of the ubiquitin/proteasome system in the initiation of negative signaling by cytokine receptors.

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and N (G)-monomethyl- l-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH 4) depletion eNOS also generates (*)O 2 (-); however, the effects of methylarginines on eNOS-derived (*)O 2 (-) generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on (*)O 2 (-) production from eNOS under conditions of BH 4 depletion. In the absence of BH 4, ADMA dose-dependently increased NOS-derived (*)O 2 (-) generation, with a maximal increase of 151% at 100 microM ADMA. L-NMMA also dose-dependently increased NOS-derived (*)O 2 (-), but to a lesser extent, demonstrating a 102% increase at 100 microM L-NMMA. Moreover, the native substrate l-arginine also increased eNOS-derived (*)O 2 (-), exhibiting a similar degree of enhancement as that observed with ADMA. Measurements of NADPH consumption from eNOS demonstrated that binding of either l-arginine or methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies suggest, just as for l-arginine and L-NMMA, the binding of ADMA shifts the eNOS heme to the high-spin state, indicative of a more positive heme redox potential, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and (*)O 2 (-) generation from eNOS. These observations have important implications with regard to the therapeutic use of l-arginine and the methylarginine-NOS inhibitors in the treatment of disease.

Expression of leucine-rich alpha2 glycoprotein (LRG), a member of the leucine-rich repeat family of proteins, was recently shown to be up-regulated during neutrophil differentiation. Its precise role in granulopoiesis, however, remains unknown. In this paper, we show that the transcription factors PU.1 and C/EBPepsilon that regulate the expression of multiple myeloid-specific genes also bind to the LRG promoter. We also demonstrate that LRG localizes to the same cytoplasmic compartment as myeloperoxidase and that G-CSF treatment of the 32Dcl3 myeloid cell line induces nuclear translocation of LRG. Stable transfection of LRG into 32Dcl3 cells resulted in accelerated, G-CSF-mediated neutrophil differentiation and induction of CD11b expression. In contrast, constitutive expression of LRG in 32Dwt18 cells, expressing a chimeric erythropoietin (Epo)/G-CSFR consisting of the EpoR extracellular domain fused to the G-CSFR transmembrane and cytoplasmic domains, failed to induce accelerated neutrophil differentiation and CD11b expression in response to Epo stimulation. LRG-mediated accelerated differentiation and CD11b expression were found to correlate with an increased level of phospho-Stat3 but not with PU.1 or p27(kip1) levels. Hence, similar to other genes involved in neutrophil differentiation, the expression of LRG also appears to be regulated by PU.1 and C/EBPepsilon. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via nonredundant G-CSFR signals.

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.

Nitric oxide synthase is inhibited by NG-methylated derivatives of arginine whose cellular levels are controlled by dimethylarginine dimethylamino-hydrolase (DDAH). DDAH-1 is a Zn(II)-containing enzyme that through hydrolysis of methylated l-arginines regulates the activity of NOS. Herein, we report the kinetic properties of hDDAH-1 and its redox-dependent regulation. Kinetic studies using recombinant enzyme demonstrated Km values of 68.7 and 53.6 microM and Vmax values of 356 and 154 nmols/mg/min for ADMA and L-NMMA, respectively. This enzymatic activity was selective for free ADMA and L-NMMA and was incapable of hydrolyzing peptide incorporated methylarginines. Subsequent studies performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching >or=100 microM were needed to confer significant inhibition of DDAH activity. However, exposure of DDAH to the lipid oxidation product, 4-HNE, dose-dependently inhibited DDAH activity with 15% inhibition observed at 10 microM, 50% inhibition at 50 microM, and complete inhibition at 500 microM. Mass spectrometry analysis demonstrated that the mechanism of inhibition resulted from the formation of Michael adducts on His 173, which lies within the active site catalytic triad of hDDAH-1. These studies were performed with pathophysiologicaly relevant concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Because DDAH is the primary enzyme involved in methylarginine metabolism, the loss of activity of this enzyme would result in impaired NOS activity and reduced NO bioavailability.

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Delta716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Delta716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation.

Coronary vasodilation is impaired in the postischemic heart with a loss of endothelial nitric oxide synthase (eNOS) activity, but the mechanisms underlying ischemia-induced eNOS dysfunction are not understood. For nitric oxide (NO) synthesis, eNOS requires the redox-sensitive cofactor tetrahydrobiopterin (BH(4)); however, the role of BH(4) in ischemia-induced endothelial dysfunction remains unknown. Therefore, isolated rat hearts were subjected to varying durations of ischemia, and the alterations in NOS-dependent vasodilation were measured and correlated with assays of eNOS activity and cardiac BH(4) concentrations. Ischemia time-dependently decreased cardiac BH(4) content with 85, 95, or 97% irreversible degradation after 30, 45, or 60 min of ischemia, respectively. Paralleling the decreases in BH(4), reductions of eNOS activity were seen of 58, 86, or 92%, and NOS-derived superoxide production was greatly increased. Addition of 10 microM BH(4) enhanced eNOS activity in nonischemic hearts and partially restored activity after ischemia. It also suppressed NOS-derived superoxide production. Impaired coronary flow during postischemic reperfusion was improved by BH(4) infusion. Thus, BH(4) depletion contributes to postischemic eNOS dysfunction, and BH(4) treatment is effective in partial restoration of endothelium-dependent coronary flow. Supplementation of BH(4) may therefore be an important therapeutic approach to reverse endothelial dysfunction in postischemic tissues.

Objective: To examine the influence of nonthermal ultrasound on mechano-growth factor (MGF) messenger ribonucleic acid (mRNA) expression after blunt trauma., Design: A 2x4 factorial multivariate analysis of variance design., Setting: University research laboratory., Animals: Thirty-six 3- to 4-month-old male Wistar rats (mean weight, 280.8+/-21.5g). Thirty-two received a bilateral contusion injury to the gastrocnemius via a drop mass technique. Four were control animals., Intervention: Ultrasound treatment (frequency, 3MHz; intensity, 0.3W/cm(2); continuous duty cycle) was started 24 hours postinjury and delivered for 5 minutes daily on 4 consecutive days. Treatment was on the left hindlimb and the contralateral right hindlimb was the nonultrasound control., Main Outcome Measures: Muscle mass (in grams) and MGF mRNA expression as measured via real-time reverse transcriptase polymerase chain reaction., Results: Ultrasound had no effect on muscle mass (F(1,28)=2.723, P=.110, 1-beta=.357, eta(2)=.089). Ultrasound treatments decreased MGF mRNA expression in the treated limb compared with the nontreated hindlimb (F(1,28)=6.605, P=.016, 1-beta=.699, eta(2)=.191)., Conclusions: The nonthermal ultrasound treatments resulted in decreased MGF mRNA expression after blunt trauma to the gastrocnemius muscles.

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.

Discoidin domain receptors (DDR1 and DDR2) are widely expressed cell-surface receptors, which bind to and are activated by collagens, including collagen type 1. Activation of DDRs and the resulting downstream signaling is known to regulate the extracellular matrix. However, little is known about how DDRs interact with collagen and its direct impact on collagen regulation. Here, we have established that by binding to collagen, the extracellular domain (ECD) of DDR2 inhibits collagen fibrillogenesis and alters the morphology of collagen type 1 fibers. Our in vitro assays utilized DDR2-Fc fusion proteins, which contain only the ECD of DDR2. Using surface plasmon resonance, we confirmed that further oligomerization of DDR2-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Collagen turbidity measurements and biochemical assays indicated that DDR2 delays the formation of collagen fibrils. Atomic force microscopy of soluble collagen revealed that a predominately monomeric state of collagen was present with DDR2, while control solutions had an abundance of polymeric collagen. Transmission electron microscopy of collagen fibers, showed that the native periodic banded structure of collagen fibers was weakened and nearly absent in the presence of DDR2. Further, using a cell-based assay we demonstrate that overexpression of full length DDR2 inhibits fibrillogenesis of collagen type 1. Our results demonstrate a novel and important functional role of the DDR2 ECD that may contribute to collagen regulation via modulation of fibrillogenesis.

Mutations in the ELA2 gene encoding neutrophil elastase (NE) are present in most patients with severe congenital neutropenia (SCN). However, the mechanisms by which these mutations cause neutropenia remain unknown. To investigate the effects of mutant NE expression on granulopoiesis, we used the HL-60 promyelocytic cell line retrovirally transduced with the G185R NE mutant that is associated with a severe SCN phenotype. We show that the mutant enzyme accelerates apoptosis of differentiating but not of proliferating cells. Using metabolic labeling, confocal immunofluorescence microscopy, and immunoblot analysis of subcellular fractions, we also demonstrate that the G185R mutant is abnormally processed and localizes predominantly to the nuclear and plasma membranes rather than to the cytoplasmic compartment observed with the wild-type (WT) enzyme. Expression of the G185R mutant appeared to alter the subcellular distribution and expression of adaptor protein 3 (AP3), which traffics proteins from the trans-Golgi apparatus to the endosome. These observations provide further insight into potential mechanisms by which NE mutations cause neutropenia and suggest that abnormal protein trafficking and accelerated apoptosis of differentiating myeloid cells contribute to the severe SCN phenotype resulting from the G185R mutation.

Severe congenital neutropenia (SCN) is a rare disease diagnosed at or soon after birth, characterized by a myeloid maturation arrest in the bone marrow, ineffective neutrophil production, and recurrent infections. Most patients respond to treatment with granulocyte colony-stimulating factor (G-CSF), and the majority harbor mutations in the neutrophil elastase gene. In the subset of patients with SCN transforming to acute myeloid leukemia (AML), mutations that truncate the cytoplasmic tail of the G-CSF receptor (G-CSFR) have been detected. Here, we report a novel mutation in the extracellular portion of the G-CSFR within the WSXWS motif in a patient with SCN without AML who was refractory to G-CSF treatment. The mutation affected a single allele and introduced a premature stop codon that deletes the distal extracellular region and the entire transmembrane and cytoplasmic portions of the G-CSFR. Expression of the mutant receptor in either myeloid or lymphoid cells was shown to alter subcellular trafficking of the wild-type (WT) G-CSFR by constitutively heterodimerizing with it. WT/mutant G-CSFR heterodimers appeared to be retained in the endoplasmic reticulum and/or Golgi and accumulate intracellularly. These findings together with 2 previous case reports of extracellular mutations in the G-CSFR in patients with SCN unresponsive to G-CSF suggest a common mechanism underlying G-CSF refractoriness.

Maintenance of p53 function is important for normal cell growth and development, and loss of p53 function contributes directly to malignant tumor development. The recently discovered Pirh2 protein is an ubiquitin-protein ligase that negatively regulates p53 through activity by targeting it for degradation. To determine how Pirh2 may mediate lung tumorigenesis, we evaluated Pirh2 expression in human and mouse lung tumor samples and paired normal lung tissues using immunoblot analysis and immunohistochemistry. Pirh2 protein expression was higher in 27 (84%) of 32 human lung neoplasms than in matched normal lung tissue and in 14 of 15 mouse lung tumors evaluated. In addition, p53 protein was more ubiquitinated in the mouse lung tumors than in normal tissue, and overall p53 expression was lower than that in normal tissue. These results are consistent with the hypothesis that increased Pirh2 expression affects lung tumorigenesis by reducing p53 activity. To our knowledge, this is the first description of altered Pirh2 expression in human and mouse tumors.

Mutations in the G-CSF receptor (G-CSFR) in patients with severe congenital neutropenia (SCN) are postulated to contribute to transformation to acute myelogenous leukemia (AML). These mutations result in defective receptor internalization and sustained cellular activation, suggesting a loss of negative signaling by the G-CSFR. In this paper we investigated the roles of SHIP and cytokine-inducible Src homology 2 protein (CIS) in down-modulating G-CSFR signals and demonstrate that loss of their recruitment as a consequence of receptor mutations leads to aberrant signaling. We show that SHIP binds to phosphopeptides corresponding to Tyr744 and Tyr764 in the G-CSFR and that Tyr764 is required for in vivo phosphorylation of SHIP and the formation of SHIP/Shc complexes. Cells expressing a G-CSFR form lacking Tyr764 exhibited hypersensitivity to G-CSF and enhanced proliferation, but to a lesser degree than observed with the most common mutant G-CSFR form in patients with SCN/AML, prompting us to investigate whether suppressor of cytokine signaling proteins also down-modulate G-CSFR signals. G-CSF was found to induce the expression of CIS and of CIS bound to phosphopeptides corresponding to Tyr729 and Tyr744 of the G-CSFR. The expression of CIS was prolonged in cells with the SCN/AML mutant G-CSFR lacking Tyr729 and Tyr744, which also correlated with increased G-CSFR expression. These findings suggest that SHIP and CIS interact with distal phosphotyrosine residues in the G-CSFR to negatively regulate G-CSFR signaling by limiting proliferation and modulating surface expression of the G-CSFR, respectively. Novel therapeutic approaches targeting inhibitory pathways that limit G-CSFR signaling may have promise in the treatment of patients with SCN/AML.