Publications by authors named "Robert P Hebbel"

98 Publications

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Front Immunol 2020 19;11:613278. Epub 2021 Jan 19.

Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, United States.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS- had similar complete blood counts and serum chemistries as SS- mice. However, SS- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS- mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS- livers, SS- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS- or SS- bone marrow into AA- or AA- recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA- . These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.
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http://dx.doi.org/10.3389/fimmu.2020.613278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851052PMC
January 2021

Targeting AnxA1/Fpr2/ALX Pathway Regulates Neutrophil Function Promoting Thrombo-Inflammation Resolution in Sickle Cell Disease.

Blood 2021 Jan 26. Epub 2021 Jan 26.

Louisiana State University Health Sciences Center, United States.

Neutrophils plays a crucial role in the intertwined processes of thrombosis and inflammation. Altered neutrophil phenotype may contribute to inadequate resolution which is known to be a major pathophysiological contributor of thrombo-inflammatory conditions such as Sickle Cell Disease (SCD). The endogenous protein Annexin A1 (AnxA1) facilitates inflammation resolution via Formyl Peptide Receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting neutrophil dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of FPR2/ALX (a fundamental receptor involved in resolution) pathway. We demonstrated direct evidence that neutrophils with SCD phenotype play a key role in contributing to thrombo-inflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. Herein, we present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thrombo-inflammatory conditions such as SCD.
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http://dx.doi.org/10.1182/blood.2020009166DOI Listing
January 2021

Relationship of Circulating Endothelial Cells With Obesity and Cardiometabolic Risk Factors in Children and Adolescents.

J Am Heart Assoc 2021 Jan 29;10(1):e018092. Epub 2020 Dec 29.

Center for Pediatric Obesity Medicine University of Minnesota Medical School Minneapolis MN.

Background Circulating endothelial cells (CECs) reflect early changes in endothelial health; however, the degree to which CEC number and activation is related to adiposity and cardiovascular risk factors in youth is not well described. Methods and Results Youth in this study (N=271; aged 8-20 years) were classified into normal weight (body mass index [BMI] percentage <85th; n=114), obesity (BMI percentage ≥95th to <120% of the 95th; n=63), and severe obesity (BMI percentage ≥120% of the 95th; n=94) catagories. CEC enumeration was determined using immunohistochemical examination of buffy coat smears and activated CEC (percentage of vascular cell adhesion molecule-1 expression) was assessed using immunofluorescent staining. Cardiovascular risk factors included measures of body composition, blood pressure, glucose, insulin, lipid profile, C-reactive protein, leptin, adiponectin, oxidized low-density lipoprotein cholesterol, carotid artery intima-media thickness, and pulse wave velocity. Linear regression models examined associations between CEC number and activation with BMI and cardiovascular risk factors. CEC number did not differ among BMI classes (>0.05). Youth with severe obesity had a higher degree of CEC activation compared with normal weight youth (8.3%; 95% CI, 1.1-15.6 [=0.024]). Higher CEC number was associated with greater body fat percentage (0.02 per percentage; 95% CI, 0.00-0.03 [=0.020]) and systolic blood pressure percentile (0.01 per percentage; 95% CI, 0.00-0.01 [=0.035]). Higher degree of CEC activation was associated with greater visceral adipose tissue (5.7% per kg; 95% CI, 0.4-10.9 [=0.034]) and non-high-density lipoprotein cholesterol (0.11% per mg/dL; 95% CI, 0.01-0.21 [=0.039]). Conclusions Methods of CEC quantification are associated with adiposity and cardiometabolic risk factors and may potentially reflect accelerated atherosclerosis as early as childhood.
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http://dx.doi.org/10.1161/JAHA.120.018092DOI Listing
January 2021

SARS-CoV-2 severity in African Americans - A role for Duffy Null?

Haematologica 2020 12 1;105(12):2892. Epub 2020 Dec 1.

Division of Hematology-Oncology-Transplantation, University of Minnesota Medical School, Minneapolis, MN 55455 USA.

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http://dx.doi.org/10.3324/haematol.2020.269415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716376PMC
December 2020

Abnormal Endothelial Gene Expression Associated With Early Coronary Atherosclerosis.

J Am Heart Assoc 2020 07 16;9(14):e016134. Epub 2020 Jul 16.

Department of Cardiovascular Diseases Mayo Clinic College of Medicine and Science Rochester MN.

Background We examined feasibility of a unique approach towards gaining insight into heritable risk for early atherosclerosis: surveying gene expression by endothelial cells from living subjects. Methods and Results Subjects aged <50 years (mean age, 37; range, 22-49) without obstructive coronary artery disease underwent coronary reactivity testing that identified them as having normal or abnormal coronary endothelial function. Cultures of Blood Outgrowth Endothelial Cells (BOEC) from 6 normal and 13 abnormal subjects passed rigorous quality control and were used for microarray assessment of gene expression. Of 9 genes differentially expressed at false discovery rate <0.1%, we here focus upon abnormal subjects having elevated expression of (high mobility group box 1) which we unexpectedly found to be linked to low (laminin gamma 1) expression. This linkage was corroborated by 3 of our past studies and confirmed bio-functionally. Compared with normal BOEC, abnormal BOEC released 13±3-fold more HMGB1 in response to lipopolysaccharide; and they deposited one tenth as much LAMC1 into collagen subendothelial matrix during culture. Clinical follow-up data are provided for 4 normal subjects (followed 13.4±0.1 year) and for 12 abnormal subjects (followed 9.1±4.5 years). Conclusions The known pathogenic effects of high- and low- predict that the combination would biologically converge upon the focal adhesion complex, to the detriment of endothelial shear responsiveness. This gene expression pattern may comprise a heritable risk state that promotes early coronary atherosclerosis. If so, the testing could be applied even in childhood, enabling early intervention. This approach offers a way to bridge the information gap between genetics and clinical phenotype.
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http://dx.doi.org/10.1161/JAHA.120.016134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660702PMC
July 2020

Blood Outgrowth Endothelial Cells as a Cellular Carrier for Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Preclinical Models of Non-Small Cell Lung Cancer.

Transl Oncol 2020 Jul 15;13(7):100782. Epub 2020 May 15.

University of Minnesota, Department of Medicine, Division of Hematology, Oncology, and Transplantation, Minneapolis, MN, USA.

Oncolytic viruses have demonstrated efficacy in numerous tumor models including non-small cell lung cancer (NSCLC). One limitation of viral therapy for metastatic lung cancer is that systemic administration can be hindered by complement and antiviral immunity. Thus, we investigated whether ex vivo-infected blood outgrowth endothelial cells (BOECs) could be used to deliver VSV-IFNβ in preclinical models of NSCLC. BOECs were obtained from human donors or C57/Bl6 mice. VSV was engineered to produce GFP or IFNβ. Human and murine BOECs could be infected by VSV-GFP and VSV-IFNβ. Infected BOECs resulted in killing of NSCLC cells in vitro and shielded VSV-IFNβ from antibody neutralization. Mouse BOECs localized to lungs of mice bearing syngeneic LM2 lung tumors, and infected murine BOECs reduced tumor burden in this model. In an immune-deficient A549 xenograft model, mice treated with VSV-IFNβ-infected human BOECs exhibited superior antitumor activity and survival of mice (n = 10, P < .05 compared to VSV-IFNβ alone). We conclude that BOECs can be used as a carrier for delivery of oncolytic VSV-IFNβ. This may be an effective strategy for clinical translation of oncolytic virotherapy for patients with metastatic NSCLC.
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http://dx.doi.org/10.1016/j.tranon.2020.100782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231872PMC
July 2020

The multifaceted role of ischemia/reperfusion in sickle cell anemia.

J Clin Invest 2020 03;130(3):1062-1072

Sickle cell anemia is a unique disease dominated by hemolytic anemia and vaso-occlusive events. The latter trigger a version of ischemia/reperfusion (I/R) pathobiology that is singular in its origin, cyclicity, complexity, instability, perpetuity, and breadth of clinical consequences. Specific clinical features are probably attributable to local I/R injury (e.g., stroke syndromes) or remote organ injury (e.g., acute chest syndrome) or the systematization of inflammation (e.g., multifocal arteriopathy). Indeed, by fashioning an underlying template of endothelial dysfunction and vulnerability, the robust inflammatory systematization no doubt contributes to all sickle pathology. In this Review, we highlight I/R-targeting therapeutics shown to improve microvascular blood flow in sickle transgenic mice undergoing I/R, and we suggest how such insights might be translated into human therapeutic strategies.
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http://dx.doi.org/10.1172/JCI133639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269579PMC
March 2020

Reproducibility of endothelial microparticles in children and adolescents.

Biomark Med 2020 01 15;14(1):43-51. Epub 2019 Nov 15.

Center for Pediatric Obesity Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

We assessed reproducibility of endothelial microparticles (EMPs) enumeration among youth. Four microparticle (MP) indices - total MP per microliter platelet free plasma (PFP), total EMPs per microliter PFP, percent activated EMPs and percent lactadherin positive (LACT[+]) of total EMPs - were measured at two visits (baseline and 7 ± 3 days follow-up) to determine reproducibility overall and by obesity status. We examined CD31 or CD144 with CD41-EMP events of size 0.3-1.0 μm. No statistically significant differences were observed between visits for any of the four MP indices. The within-participant and between-participant coefficient of variation was acceptable (range: 1.13-2.37) with good intraclass-correlation coefficient for all indices except total MP per microliter (range: 0.10-1.00). Total EMPs per microliter PFP, percent-activated EMPs and percent LACT(+) of total EMPs are reproducible among youth.
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http://dx.doi.org/10.2217/bmm-2019-0229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202266PMC
January 2020

Blood outgrowth endothelial cells overexpressing eNOS mitigate pulmonary hypertension in rats: a unique carrier cell enabling autologous cell-based gene therapy.

Transl Res 2019 08 24;210:1-7. Epub 2019 Apr 24.

Division of Hematology-Oncology-Transplantation, Department of Medicine; and Vascular Biology Center, University of Minnesota Medical School, Minneapolis, Minnesota.

We have investigated a unique cell type, blood outgrowth endothelial cells (BOEC), as a cell-based gene therapy approach to pulmonary hypertension. BOEC are bona fide endothelial cells, obtained from peripheral blood, that can be expanded to vast numbers, and are amenable to both cryopreservation and genetic modification. We established primary cultures of rat BOEC and genetically altered them to over-express human eNOS plus green fluorescent protein (rBOEC/eNOS) or to express GFP only (rBOEC/GFP). We gave monocrotaline to rats on day 0, and they developed severe pulmonary hypertension. As a Prevention model, we infused saline or rBOEC/GFP or rBOEC/eNOS on day 3, and then examined endpoints on day 24. The rBOEC/eNOS recipients developed elevated NOx (serum and lung) and less severe: elevation of right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary arteriolar muscularization and loss of alveolar density. As an Intervention model, we waited until day 21 to give the test infusions, and we examined endpoints on day 35. The rBOEC/eNOS recipients again developed elevated NOx and manifested the same improvements. Indeed, rBOEC/eNOS infusion not only prevented worsening of RVSP but also partially reversed established arteriolar muscularization. These data suggest that BOEC may be useful as a carrier cell for genetic strategies targeting pulmonary hypertension. Their properties render BOEC amenable to preclinical and scale-up studies, available for autologous therapies, and tolerant of modification and storage for potential future use in patients at risk for PAH, eg, as defined by genetics or medical condition.
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http://dx.doi.org/10.1016/j.trsl.2019.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6741773PMC
August 2019

Morphine promotes neovascularizing retinopathy in sickle transgeneic mice.

Blood Adv 2019 04;3(7):1073-1083

Vascular Biology Center, and.

Neovascularizing retinopathy is a significant complication of sickle cell disease (SCD), occurring more frequently in HbSC than HbSS disease. This risk difference is concordant with a divergence of angiogenesis risk, as identified by levels of pro- vs anti-angiogenic factors in the sickle patient's blood. Because our prior studies documented that morphine promotes angiogenesis in both malignancy and wound healing, we tested whether chronic opioid treatment would promote retinopathy in NY1DD sickle transgenic mice. After 10 to 15 months of treatment, sickle mice treated with morphine developed neovascularizing retinopathy to a far greater extent than either of the controls (sickle mice treated with saline and wild-type mice treated identically with morphine). Our dissection of the mechanistic linkage between morphine and retinopathy revealed a complex interplay among morphine engagement with its μ opioid receptor (MOR) on retinal endothelial cells (RECs); morphine-induced production of tumor necrosis factor α and interleukin-6 (IL-6), causing increased expression of both MOR and vascular endothelial growth factor receptor 2 (VEGFR2) on RECs; morphine/MOR engagement transactivating VEGFR2; and convergence of MOR, VEGFR2, and IL-6 activation on JAK/STAT3-dependent REC proliferation and angiogenesis. In the NY1DD mice, the result was increased angiogenesis, seen as neovascularizing retinopathy, similar to the retinal pathology occurring in humans with SCD. Therefore, we conclude that chronic opioid exposure, superimposed on the already angiogenic sickle milieu, might enhance risk for retinopathy. These results provide an additional reason for development and application of opioid alternatives for pain control in SCD.
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http://dx.doi.org/10.1182/bloodadvances.2018026898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457224PMC
April 2019

The missing middle of sickle therapeutics: Multi-agent therapy, targeting risk, using biomarkers.

Am J Hematol 2018 12 23;93(12):1439-1443. Epub 2018 Oct 23.

Division of Hematology/Oncology, Department of Medicine, Augusta University, Augusta, Georgia.

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http://dx.doi.org/10.1002/ajh.25289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283073PMC
December 2018

Sickle hemoglobin oxygen affinity-shifting strategies have unequal cerebrovascular risks.

Am J Hematol 2018 03 6;93(3):321-325. Epub 2017 Dec 6.

Hematology-Oncology-Transplantation, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, 555455.

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http://dx.doi.org/10.1002/ajh.24975DOI Listing
March 2018

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Am J Hematol 2017 Nov 29;92(11):1119-1130. Epub 2017 Jul 29.

Division of Hematology-Oncology-Transplantation, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+S , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.
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http://dx.doi.org/10.1002/ajh.24856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655742PMC
November 2017

Blood endothelial cells: utility from ambiguity.

Authors:
Robert P Hebbel

J Clin Invest 2017 May 1;127(5):1613-1615. Epub 2017 May 1.

In the mid-1990s, my research group began to devise a method to establish endothelial cell cultures from human peripheral blood, with an ultimate goal of examining interindividual heterogeneity of endothelial biology. The initial work, published in the JCI in 2000, described the method enabling successful attainment of blood outgrowth endothelial cells (BOEC). Truly endothelial, BOEC are progeny of a transplantable cell that originates in bone marrow, a putative endothelial progenitor. Our subsequent experimental work focused upon practical applications of BOEC: their use for gene therapy, tissue engineering, assessment of mutant gene effect, and discovery of heterogeneity in endothelial biology.
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http://dx.doi.org/10.1172/JCI93649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409783PMC
May 2017

Microparticles in sickle cell anaemia: promise and pitfalls.

Br J Haematol 2016 07 2;174(1):16-29. Epub 2016 May 2.

Division of Haematology and Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Blood from patients with sickle cell disease contains microparticles (MP) derived from multiple cell sources, including red cells, platelets, monocytes and endothelial cells. MPs are of great interest because of their disease associations, their status as promising biomarkers, and the intercellular communications they mediate. To illustrate the likelihood of their relevance in sickle cell disease, we discuss the nature of MP, their profiling in sickle disease, some caveats relevant to their detection, their roles in supporting coagulation and the disparate influences they may exert upon the pathobiology of sickle cell disease.
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http://dx.doi.org/10.1111/bjh.14112DOI Listing
July 2016

Reproducibility of circulating endothelial cell enumeration and activation in children and adolescents.

Biomark Med 2016 05 13;10(5):463-71. Epub 2016 Apr 13.

Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

Introduction: We examined the reproducibility of circulating endothelial cells (CEC) enumeration and activation among youth.

Materials And Methods: CECs from 151 youth were measured at baseline and 1 week follow-up. Enumeration of CEC in fresh whole blood was determined by direct assessment of buffy coat smears (CD146+ nucleated cells) and activated CEC (%VCAM-1 expression) was determined after immunomagnetic enrichment and co-staining of nuclei, plus positivity for P1H12 and VCAM-1.

Results: No statistically significant difference in CEC enumeration (1.2 ± 2.5 vs 1.3 ± 2.2 CEC/milliliter of whole blood, p = 0.745) or activated CEC (57.1 ± 24.4 vs 58.0 ± 21.3 %VCAM-1, p = 0.592) between baseline and 1 week follow-up.

Conclusion: On a cohort basis, CEC enumeration and activation are reproducible in youth. Relatively high individual biological variability may limit its clinical utility.
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http://dx.doi.org/10.2217/bmm-2015-0051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827818PMC
May 2016

Sickle cell disease: renal manifestations and mechanisms.

Nat Rev Nephrol 2015 Mar 10;11(3):161-71. Epub 2015 Feb 10.

Division of Haematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Mayo Mail Code 480, 420 Delaware Street S. E., Minneapolis, MN 55455, USA.

Sickle cell disease (SCD) substantially alters renal structure and function, and causes various renal syndromes and diseases. Such diverse renal outcomes reflect the uniquely complex vascular pathobiology of SCD and the propensity of red blood cells to sickle in the renal medulla because of its hypoxic, acidotic, and hyperosmolar conditions. Renal complications and involvement in sickle cell nephropathy (SCN) include altered haemodynamics, hypertrophy, assorted glomerulopathies, chronic kidney disease, acute kidney injury, impaired urinary concentrating ability, distal nephron dysfunction, haematuria, and increased risks of urinary tract infections and renal medullary carcinoma. SCN largely reflects an underlying vasculopathy characterized by cortical hyperperfusion, medullary hypoperfusion, and an increased, stress-induced vasoconstrictive response. Renal involvement is usually more severe in homozygous disease (sickle cell anaemia, HbSS) than in compound heterozygous types of SCD (for example HbSC and HbSβ(+)-thalassaemia), and is typically mild, albeit prevalent, in the heterozygous state (sickle cell trait, HbAS). Renal involvement contributes substantially to the diminished life expectancy of patients with SCD, accounting for 16-18% of mortality. As improved clinical care promotes survival into adulthood, SCN imposes a growing burden on both individual health and health system costs. This Review addresses the renal manifestations of SCD and focuses on their underlying mechanisms.
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http://dx.doi.org/10.1038/nrneph.2015.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701210PMC
March 2015

H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice.

Front Pharmacol 2014 17;5:79. Epub 2014 Apr 17.

Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN USA ; Vascular Biology Center, Department of Medicine, University of Minnesota Medical School Minneapolis, MN, USA.

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe(2+) from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes Fe(2+) to catalytically inactive Fe(3+) inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic SCD mice in response to plasma hemoglobin. We utilized a Sleeping Beauty (SB) transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections into NY1DD SCD mice. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or a human triple missense FHC (ms-hFHC) plasmid with no ferroxidase activity. 8 weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) 1 h after infusion of stroma-free hemoglobin, while mice overexpressing wt-hFHC had only 5% stasis (p < 0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, ferritin light chain (FLC), 5-aminolevulinic acid synthase (ALAS), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 activity and protein. There was also a decrease in hepatic activated nuclear factor-kappa B (NF-κB) phospho-p65 and vascular cell adhesion molecule-1 (VCAM-1). Inhibition of HO-1 activity with tin protoporphyrin demonstrated HO-1 was not essential for the protection by wt-hFHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, adhesion molecules, and microvascular stasis in transgenic SCD mice.
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http://dx.doi.org/10.3389/fphar.2014.00079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029007PMC
May 2014

Ischemia-reperfusion injury in sickle cell anemia: relationship to acute chest syndrome, endothelial dysfunction, arterial vasculopathy, and inflammatory pain.

Authors:
Robert P Hebbel

Hematol Oncol Clin North Am 2014 Apr;28(2):181-98

Division of Hematology-Oncology-Transplantation, Department of Medicine, University of Minnesota Medical School, 420 Delaware Street South East, Mayo Mail Code 480, Minneapolis, MN 55455, USA. Electronic address:

Ischemia-reperfusion (I/R) physiology, also called reperfusion injury, instigates vascular and tissue injury in human disease states. This review describes why sickle cell anemia should be conceptualized in this fashion and how I/R physiology explains the genesis of characteristic aspects of vascular pathobiology and clinical disease in sickle cell anemia. The nature of I/R and its relevance to sickle cell anemia are discussed, with an emphasis on the acute chest syndrome, endothelial dysfunction with aberrant vasoregulation, circle of Willis vasculopathy, and inflammatory pain. Viewing sickle disease from this perspective elucidates defining pathophysiology and identifies a host of novel potential therapeutic targets.
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http://dx.doi.org/10.1016/j.hoc.2013.11.005DOI Listing
April 2014

Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease.

Blood 2014 Mar 21;123(11):1747-56. Epub 2014 Jan 21.

Division of Hematology/Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC;

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.
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http://dx.doi.org/10.1182/blood-2013-08-523936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954055PMC
March 2014

Blood outgrowth endothelial cells alter remodeling of completely biological engineered grafts implanted into the sheep femoral artery.

J Cardiovasc Transl Res 2014 Mar 16;7(2):242-9. Epub 2014 Jan 16.

Department of Biomedical Engineering, University of Minnesota, 7-114 NHH, 312 Church St SE, Minneapolis, MN, 55455, USA.

Hemocompatibility of tissue-engineered vascular grafts remains a major hurdle to clinical utility for small-diameter grafts. Here we assessed the feasibility of using autologous blood outgrowth endothelial cells to create an endothelium via lumenal seeding on completely biological, decellularized engineered allografts prior to implantation in the sheep femoral artery. The 4-mm-diameter, 2- to 3-cm-long grafts were fabricated from fibrin gel remodeled into an aligned tissue tube in vitro by ovine dermal fibroblasts prior to decellularization. Decellularized grafts pre-seeded with blood outgrowth endothelial cells (n = 3) retained unprecedented (>95 %) monolayer coverage 1 h post-implantation and had greater endothelial coverage, smaller wall thickness, and more basement membrane after 9-week implantation, including a final week without anti-coagulation therapy, compared with contralateral non-seeded controls. These results support the use of autologous blood outgrowth endothelial cells as a viable source of endothelial cells for creating an endothelium with biological function on decellularized engineered allografts made from fibroblast-remodeled fibrin.
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http://dx.doi.org/10.1007/s12265-013-9539-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213739PMC
March 2014

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

Blood 2014 Jan 25;123(3):377-90. Epub 2013 Nov 25.

Division of Hematology, Oncology and Transplantation, Vascular Biology Center, University of Minnesota, Minneapolis, MN;

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.
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http://dx.doi.org/10.1182/blood-2013-04-495887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894494PMC
January 2014

Pilot study of vascular health in survivors of osteosarcoma.

Pediatr Blood Cancer 2013 Oct 30;60(10):1703-8. Epub 2013 May 30.

Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Background: Cardiovascular-related toxicities have been reported among survivors of osteosarcoma.

Methods: Fasting blood samples from 24 osteosarcoma survivors were analyzed for high-sensitivity C-reactive protein (hsCRP), triglycerides, total cholesterol, high-density lipoprotein (HDL), apolipoprotein-ß, lipoprotein (a), fibrinogen, circulating endothelial cells (CECs), and surface expression of vascular cell adhesion molecule-1 (VCAM-1). Values were compared to subjects in the natural history Coronary Artery Risk Development in Young Adults (CARDIA) cohort study except for CECs and VCAM-1 expression, which were compared to controls studied at the University of Minnesota Lillehei clinical trials unit.

Procedure: Survivors (54.2% male), median age 18 years (9-32) at diagnosis, 36.5 years (20-56) at evaluation were treated with a variety of chemotherapeutic exposures, all but one were exposed to doxorubicin (median dose 450 mg/m(2) ; range: 90-645 mg/m(2)), 14 (58.3%) received cisplatin, and 3 (12.5%) were exposed to carboplatin. Two survivors (8.3%) received radiation therapy for disease relapse. Compared to CARDIA subjects, mean hsCRP (3.0 mg/L ± 2.0 vs. 1.6 ± 2.3), triglycerides (151 mg/dl ± 81.7 vs. 95.4 ± 101.3), lipoprotein (a) (34.9 mg/dl ± 17.7 vs. 13.8 ± 22.0), and fibrinogen (315.0 mg/dl ± 49.3 vs. 252.4 ± 61.7) were significantly elevated. The number of CECs (0.47 cells/ml ± 2.5 vs. 0.92 ± 2.5) did not differ while surface expression of VCAM-1 (86.4% ± 34.0 vs. 42.1 ± 33.8) was significantly elevated compared to controls.

Conclusions: Among survivors of osteosarcoma, assessed a median of 14 years from diagnosis, there is evidence of vascular inflammation, dyslipidemia, and early atherogenesis.
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http://dx.doi.org/10.1002/pbc.24610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968821PMC
October 2013

A mechanistic role for DNA methylation in endothelial cell (EC)-enriched gene expression: relationship with DNA replication timing.

Blood 2013 Apr 28;121(17):3531-40. Epub 2013 Feb 28.

Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.

Proximal promoter DNA methylation has been shown to be important for regulating gene expression. However, its relative contribution to the cell-specific expression of endothelial cell (EC)-enriched genes has not been defined. We used methyl-DNA immunoprecipitation and bisulfite conversion to analyze the DNA methylation profile of EC-enriched genes in ECs vs nonexpressing cell types, both in vitro and in vivo. We show that prototypic EC-enriched genes exhibit functional differential patterns of DNA methylation in proximal promoter regions of most (eg, CD31, von Willebrand factor [vWF], VE-cadherin, and intercellular adhesion molecule-2), but not all (eg, VEGFR-1 and VEGFR-2), EC-enriched genes. Comparable findings were evident in cultured ECs, human blood origin ECs, and murine aortic ECs. Promoter-reporter episomal transfection assays for endothelial nitric oxide synthase, VE-cadherin, and vWF indicated functional promoter activity in cell types where the native gene was not active. Inhibition of DNA methyltransferase activity indicated important functional relevance. Importantly, profiling DNA replication timing patterns indicated that EC-enriched gene promoters with differentially methylated regions replicate early in S-phase in both expressing and nonexpressing cell types. Collectively, these studies highlight the functional importance of promoter DNA methylation in controlling vascular EC gene expression.
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http://dx.doi.org/10.1182/blood-2013-01-479170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637020PMC
April 2013

Age sensitizes the kidney to heme protein-induced acute kidney injury.

Am J Physiol Renal Physiol 2013 Feb 28;304(3):F317-25. Epub 2012 Nov 28.

Division of Nephrology and Hypertension, Mayo Clinic, Guggenheim 542, 200 First St., SW, Rochester, MN 55905, USA.

Age increases the risk for ischemic acute kidney injury (AKI). We questioned whether a similar age-dependent injury occurs following exposure to hemoglobin, a known nephrotoxin. Old mice (~16 mo old), but not young mice (~6 mo old), when administered hemoglobin, exhibited marked elevation in blood urea nitrogen (BUN) and serum creatinine, and acute tubular necrosis with prominent tubular cast formation. The aged kidney exhibited induction of heme oxygenase-1 (HO-1) and other genes/proteins that may protect against heme-mediated renal injury, including ferritin, ferroportin, haptoglobin, and hemopexin. Old mice did not evince induction of HO-2 mRNA by hemoglobin, whereas a modest induction of HO-2 mRNA was observed in young mice. To determine the functional significance of HO-2 in heme protein-induced AKI, we administered hemoglobin to relatively young HO-2(+/+) and HO-2(-/-) mice: HO-2(-/-) mice, compared with HO-2(+/+) mice, exhibited greater renal dysfunction and histologic injury when administered hemoglobin. In addition to failing to elicit a protective system such as HO-2 in response to hemoglobin, old mice exhibited an exaggerated maladaptive response typified by markedly greater induction of the nephrotoxic cytokine IL-6 (130-fold increase vs. 10-fold increase in mRNA in young mice). We conclude that aged mice, unlike relatively younger mice, are exquisitely sensitive to the nephrotoxicity of hemoglobin, an effect attended by a failure to induce HO-2 mRNA and a fulminant upregulation of IL-6. Age thus markedly augments the sensitivity of the kidney to heme proteins, and HO-2 confers resistance to such insults.
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http://dx.doi.org/10.1152/ajprenal.00606.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566520PMC
February 2013

Establishment of outgrowth endothelial cells from peripheral blood.

Nat Protoc 2012 Sep 23;7(9):1709-15. Epub 2012 Aug 23.

Department of Plasma Proteins, Landsteiner Laboratory, Academic Medical Centre and Sanquin, University of Amsterdam, Amsterdam, The Netherlands.

Blood outgrowth endothelial cells (BOECs) are important tools when investigating diagnostic and therapeutic approaches for vascular disease. In this protocol, mononuclear cells are isolated from peripheral blood and plated on type I collagen at ∼135,000 cells per cm(2) in endothelial cell differentiation medium. On average, 0.34 colonies of endothelial cells per milliliter of blood can be obtained. Colonies of endothelial cells become visible after 14-28 d. Upon confluence, these rapidly expanding colonies can be passaged and have been shown to propagate up to 10(18)-fold. Isolated BOECs are phenotypically similar to vascular endothelial cells, as revealed by their cobblestone morphology, the presence of endothelial cell-specific Weibel-Palade bodies and the expression of endothelial cell markers such as VE-cadherin. The protocol presented here also provides a particularly useful tool for the ex vivo assessment of endothelial cell function from patients with different vascular abnormalities.
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http://dx.doi.org/10.1038/nprot.2012.093DOI Listing
September 2012

Morphine promotes renal pathology in sickle mice.

Int J Nephrol Renovasc Dis 2012 20;5:109-18. Epub 2012 Jul 20.

Division of Renal Diseases and Hypertension, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA.

Patients with sickle cell disease (SCD) are often treated with opioids for severe pain. Although opioids are known to have renal-specific effects, their role in nephropathy in SCD remains unknown. Because a subset of patients receives opioids for long periods of time, we examined the influence of chronic morphine treatment on mice with pre-existing renal disease expressing varying amounts of sickle hemoglobin. Morphine treatment for 3-6 weeks resulted in a variety of defects in renal morphology observed using light and electron microscopy. Notably, morphine induced glomerular pathology, resulting in increased glomerular volume, mesangial expansion, mesangial cell proliferation, parietal cell metaplasia, podocyte effacement, and microvillus transformation. Cystic tubulopathy and hemeoxygenase-1 expression and activity were also increased in morphine-treated mice. Naloxone, a non-selective opioid receptor (OR) antagonist, ameliorated these effects. Functionally, the urine albumin to creatinine ratio was increased following acute as well as chronic morphine treatment. These results suggest that clinically relevant doses of morphine induce renal pathology and that OR antagonists may be effective for ameliorating morphine-induced renal disease.
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http://dx.doi.org/10.2147/IJNRD.S33813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413037PMC
October 2012

Pilot study of vascular health in survivors of Hodgkin lymphoma.

Pediatr Blood Cancer 2012 Aug 27;59(2):285-9. Epub 2012 Mar 27.

School of Public Health, Masonic Cancer Center, and University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Background: Vascular-related toxicities have been reported among survivors of Hodgkin lymphoma (HL), but their genesis is not well understood.

Procedure: Fasting blood samples from 25 previously irradiated HL survivors were analyzed for biomarkers that can reveal underlying inflammation and/or endothelial cell activation: high-sensitivity C-reactive protein (hsCRP), triglycerides, total cholesterol, high-density lipoprotein (HDL), apolipoprotein ß, lipoprotein (a), fibrinogen, circulating endothelial cells (CECs), and vascular cell adhesion molecule-1 (VCAM-1) expression. Values were compared to subjects in the Coronary Artery Risk Development in Young Adults (CARDIA) study. CECs and VCAM-1 were compared to healthy controls.

Results: Survivors (76% male), median age 17.6 years (5-33) at diagnosis, 33.0 years (19-55) at follow-up, included stages IA (n = 6), IIA (n = 10), IIB (n = 2), IIIA (n = 4), and IVA (n = 3) patients. Twenty-four received at least chest radiation therapy (RT) (median dose 3,150 cGy; range: 175-4,650 cGy), one received neck only; 14 (56%) had a history of anthracycline exposure (median dose: 124 mg/m(2) range: 63-200 mg/m2). Compared to CARDIA subjects, mean hsCRP (3.0 mg/L ± 2.0 vs. 1.6 ± 1.9), total cholesterol (194.1 mg/dl ± 33.2 vs. 179.4 ± 32.9), lipoprotein (a) (34.2 mg/dl ± 17.5 vs. 13.8 ± 17.5), and fibrinogen (342.0 mg/dl ± 49.1 vs. 252.6 ± 48.4) were significantly elevated. CECs (2.3 cells/ml ± 1.5 vs. 0.34 ± 1.4) were significantly elevated compared to controls. No difference in VCAM-1 expression (51.1% ± 36.8 vs. 42.3 ± 35.6) was detected.

Conclusion: HL survivors exposed to RT have evidence of vascular inflammation, dyslipidemia, and injury suggestive of early atherogenesis.
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http://dx.doi.org/10.1002/pbc.24082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3374066PMC
August 2012

Carbon-fiber microelectrode amperometry reveals sickle-cell-induced inflammation and chronic morphine effects on single mast cells.

ACS Chem Biol 2012 Mar 23;7(3):543-51. Epub 2012 Jan 23.

Department of Chemistry, Division of Hematology, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Sickle cell disease, caused by a mutation of hemoglobin, is characterized by a complex pathophysiology including an important inflammatory component. Mast cells are tissue-resident leukocytes known to influence a range of immune functions in a variety of different ways, largely through the secretion of biologically active mediators from preformed granules. However, it is not understood how mast cells influence the inflammatory environment in sickle cell disease. A notable consequence of sickle cell disease is severe pain. Therefore, morphine is often used to treat this disease. Because mast cells express opioid receptors, it is pertinent to understand how chronic morphine exposure influences mast cell function and inflammation in sickle cell disease. Herein, carbon-fiber microelectrode amperometry (CFMA) was used to monitor the secretion of immunoactive mediators from single mast cells. CFMA enabled the detection and quantification of discrete exocytotic events from single mast cells. Mast cells from two transgenic mouse models expressing human sickle hemoglobin (hBERK1 and BERK) and a control mouse expressing normal human hemoglobin (HbA-BERK) were monitored using CFMA to explore the impact of sickle-cell-induced inflammation and chronic morphine exposure on mast cell function. This work, utilizing the unique mechanistic perspective provided by CFMA, describes how mast cell function is significantly altered in hBERK1 and BERK mice, including decreased serotonin released compared to HbA-BERK controls. Furthermore, morphine was shown to significantly increase the serotonin released from HbA-BERK mast cells and demonstrated the capacity to reverse the observed sickle-cell-induced changes in mast cell function.
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http://dx.doi.org/10.1021/cb200347qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306545PMC
March 2012

Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

PLoS One 2011 28;6(12):e29110. Epub 2011 Dec 28.

Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029110PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247234PMC
May 2012