Hydroxyurea (HU) induces fetal hemoglobin synthesis through activation of cyclic guanine monophosphate (cGMP) signaling. Studies in sickle cell patients demonstrated increased circulating nitric ...oxide (NO) levels after oral HU treatment. However, the direct measurement of NO in erythroid cells and its role in fetal hemoglobin induction have not been defined. Therefore, we quantified the level of nitrate and nitrite (NOx) generated by HU in human erythroid progenitors in the presence of three nitric oxide synthase inhibitors (NOS), including N(G)-monomethyl-L-arginine (L-NMMA). In addition, cGMP levels were measured in the presence or absence of the pathway inhibitor 1H-(1,2,4)ox-adiazolo(4,3-a)quinoxalin-1-one, which blocks soluble guanylyl cyclase formation. HU treatment increased NOx levels and gamma-globin transcription in K562 and primary erythroid cells, which was augmented when HU was combined with L-NMMA. Pretreatment with the cGMP pathway inhibitor reversed gamma-gene activation by HU. These data demonstrate the direct stimulation of cellular NO and cGMP signaling in erythroid progenitors by HU as a possible mechanism for gamma-globin gene activation.
The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) ...bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.
In this review, we provide a description of those candidate biomarkers which have been demonstrated by multiple-omics approaches to vary in correlation with specific clinical manifestations of sickle ...cell severity. We believe that future clinical analyses of severity phenotype will require a multiomic analysis, or an omics stack approach, which includes integrated interactomics. It will also require the analysis of big data sets. These candidate biomarkers, whether they are individual or panels of functionally linked markers, will require future validation in large prospective and retrospective clinical studies. Once validated, the hope is that informative biomarkers will be used for the identification of individuals most likely to experience severe complications, and thereby be applied for the design of patient-specific therapeutic approaches and response to treatment. This would be the beginning of precision medicine for sickle cell disease.
Cathepsins K and V are powerful elastases elevated in endothelial cells by tumor necrosis factor-α (TNFα) stimulation and disturbed blood flow both of which contribute to inflammation-mediated ...arterial remodeling. However, mechanisms behind endothelial cell integration of biochemical and biomechanical cues to regulate cathepsin production are not known. To distinguish these mechanisms, human aortic endothelial cells (HAECs) were stimulated with TNFα and exposed to pro-remodeling or vasoprotective shear stress profiles. TNFα upregulated cathepsin K via JNK/c-jun activation, but vasoprotective shear stress inhibited TNFα-stimulated cathepsin K expression. JNK/c-jun were still phosphorylated, but cathepsin K mRNA levels were significantly reduced to almost null indicating separate biomechanical regulation of cathepsin K by shear stress separate from biochemical stimulation. Treatment with Bay 11-7082, an inhibitor of IκBα phosphorylation, was sufficient to block induction of cathepsin K by both pro-remodeling shear stress and TNFα, implicating NF-κB as the biomechanical regulator, and its protein levels were reduced in HAECs by vasoprotective shear stress. In conclusion, NF-κB and AP-1 activation were necessary to activate cathepsin K expression in endothelial cells, highlighting integration of biochemical and biomechanical stimuli to control cathepsins K and V, powerful elastases implicated for arterial remodeling due to chronic inflammation and disturbed blood flow.
Summary
The SP1/Krüppel‐like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. ...The discovery of KLF1 (EKLF), a key regulator of HBB (β‐globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ‐globin) regulation by KLF4. siRNA‐mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ‐CACCC and β‐CACCC probes demonstrated KLF4 preferentially binds the endogenous γ‐CACCC, while CREB binding protein (CREBBP) binding was not selective. Co‐immunoprecipitation studies confirmed protein‐protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co‐localization of both factors in the γ‐CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans‐activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans‐factors in HBG regulation.
In this perspective, we describe our experience as women of color scientists from diverse backgrounds and similar struggles embarking upon the National Heart, Lung and Blood Institute-funded program ...called PRIDE (Programs to Increase Diversity among Underrepresented Minorities Engaged in Health-Related Research). Under the leadership of our mentor and friend, Betty Pace, MD, a renowned and successful African American physician-scientist, the PRIDE Program was designed to address the difficulties experienced by junior-level minority investigators in establishing independent research programs and negotiating tenure and full professor status at academic institutions. The strength of PRIDE’s innovative formula was pairing us with external senior mentors and, importantly, allowing us to serve as peer mentors to each other. We believe this “Sister’s Keeper” paradigm is one solution for women to overcome their limitations and extend understandings and best practices worldwide for science, medicine, and global health.
To reduce respondent burden for future evaluations of the National Heart, Lung, and Blood Institute-supported Programs to Increase Diversity Among Individuals Engaged in Health-Related Research ...(PRIDE), a mentored-research education program, we sought to shorten the 33-item Ragins and McFarlin Mentor Role Instrument (RMMRI), measuring mentor-role appraisals, and the 69-item Clinical Research Appraisal Inventory (CRAI), measuring research self-efficacy.
Three nationally recruited, junior-faculty cohorts attended two, annual 2-3 week Summer Institutes (SI-1/SI-2: 2011/2012, 2012/2013, 2013/2014) at one of six PRIDE sites. Mentees completed the RMMRI two months after mentor assignment and the CRAI at baseline (pre-SI-1) and 6-month (mid-year) and 12-month (post-SI-2) follow-up. Publications data obtained from Scopus in October 2015 were verified with mentees' curriculum vitae. The RMMRI and CRAI were shortened using an iterative process of principal-components analysis. The shortened measures were examined in association with each other (multiple linear regression) and with increase in publications (repeated-measures analysis of covariance).
PRIDE enrolled 152 mentees (70% women; 60% Black, 35% Hispanic/Latino). Cronbach's alphas for the new 9-item RMMRI, 19-item CRAI, and four CRAI-19 subscales were excellent. Controlling for baseline self-efficacy and cohort, RMMRI-9 scores were independently, positively associated with post-SI-2 scores on the CRAI-19 and three subscales (writing, study design/data analysis, and collaboration/grant preparation). Controlling for cohort, higher RMMRI-9 and post-SI-2 CRAI-19 scores were each associated with greater increase in publications.
The RMMRI-9 and CRAI-19 retained the excellent psychometric properties of the longer measures. Findings support use of the shortened measures in future evaluations of PRIDE.
Children with hemoglobin SC (HbSC) disease suffer a significant incidence of silent cerebral infarcts but stroke is rare. A 2-year-old African American boy with HbSC disease presented with focal ...neurologic deficits associated with magnetic resonance imaging evidence of cerebral infarction with vascular abnormalities. After the acute episode he was treated with monthly transfusions and subsequently transitioned to hydroxyurea therapy. The benefits of hydroxyurea as a fetal hemoglobin inducer in HbSC disease, to ameliorate clinical symptoms are supported by retrospective studies. This case highlights the rare occurrence of stroke in a child with HbSC disease and the use of hydroxyurea therapy.
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