Publications by authors named "David A Ingram"

81 Publications

Early administration of imatinib mesylate reduces plexiform neurofibroma tumor burden with durable results after drug discontinuation in a mouse model of neurofibromatosis type 1.

Pediatr Blood Cancer 2020 08 27;67(8):e28372. Epub 2020 May 27.

Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana.

Background: Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored.

Procedure: We evaluated the effect of imatinib dosed at 10-100 mg/kg/day for 12 weeks to one-month-old Nf1 ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden.

Results: Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10 mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100 mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control.

Conclusions: Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.
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http://dx.doi.org/10.1002/pbc.28372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516834PMC
August 2020

Neurofibromin Deficiency Induces Endothelial Cell Proliferation and Retinal Neovascularization.

Invest Ophthalmol Vis Sci 2018 05;59(6):2520-2528

Department of Pediatrics and Neonatal-Perinatal Medicine, Augusta University, Augusta, Georgia, United States.

Purpose: Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization.

Methods: Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching.

Results: Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas.

Conclusions: These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial.
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http://dx.doi.org/10.1167/iovs.17-22588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963003PMC
May 2018

Adult venous endothelium is a niche for highly proliferative and vasculogenic endothelial colony-forming cells.

J Vasc Surg 2017 12 24;66(6):1854-1863. Epub 2017 Jun 24.

Health Center for Aortic Disease, Indiana University Health Center for Aortic Disease, Indianapolis, Ind; Department of Cellular and Integrative Physiology, Indiana University Health Center for Aortic Disease, Indianapolis, Ind; Department of Surgery, Indiana University Health Center for Aortic Disease, Indianapolis, Ind; Indiana Center for Vascular Biology and Medicine, Indiana University Health Center for Aortic Disease, Indianapolis, Ind.

Objective: Postnatal resident endothelium of blood vessels has been proposed to represent terminally differentiated tissue that does not replicate. We previously isolated endothelial colony-forming cells (ECFCs) from human umbilical cord blood (CB) and term placenta by using colony-forming assays and immunocytochemistry. We showed that ECFCs are highly proliferative and form functioning vessels in vivo, the defining characteristics of a true endothelial progenitor cell. This exploratory investigation was conducted to determine whether the endothelium of healthy adult blood vessels contained resident ECFCs.

Methods: The endothelium of great saphenous vein (GSV) obtained from vein stripping procedures was collected with mechanical scraping, and ECFCs were isolated according to established protocols.

Results: GSV ECFCs incorporated acetylated low-density lipoprotein, formed tubules in Matrigel (BD Biosciences, San Jose, Calif) at 24 hours, and expressed endothelial antigens cluster of differentiation (CD) 144, CD31, CD105, and kinase insert domain receptor but not hematopoietic antigen CD45. Using cumulative population doublings and single-cell assays, we demonstrated that GSV ECFCs exhibited comparable proliferative capacities compared with CB ECFCs, including similar numbers of highly proliferative cells. When injected in collagen/fibronectin gels implanted in nonobese diabetic/severe combined immune deficiency mice, GSV ECFCs formed blood vessels with circulating murine red blood cells, demonstrating their vasculogenic potential.

Conclusions: The ECFCs of the GSV contain a hierarchy of progenitor cells with a comparable number of highly proliferative clones as ECFCs of CB. The results of this investigation demonstrate that the adult endothelium contains resident progenitor cells that may have a critical role in vascular homeostasis and repair and could potentially be used as a source of autologous cells for cell therapies focusing on vasculogenesis.
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http://dx.doi.org/10.1016/j.jvs.2016.11.059DOI Listing
December 2017

Antenatal Determinants of Bronchopulmonary Dysplasia and Late Respiratory Disease in Preterm Infants.

Am J Respir Crit Care Med 2017 08;196(3):364-374

1 Pediatric Heart Lung Center, Department of Pediatrics, Children's Hospital Colorado.

Rationale: Mechanisms contributing to chronic lung disease after preterm birth are incompletely understood.

Objectives: To identify antenatal risk factors associated with increased risk for bronchopulmonary dysplasia (BPD) and respiratory disease during early childhood after preterm birth, we performed a prospective, longitudinal study of 587 preterm infants with gestational age less than 34 weeks and birth weights between 500 and 1,250 g.

Methods: Data collected included perinatal information and assessments during the neonatal intensive care unit admission and longitudinal follow-up by questionnaire until 2 years of age.

Measurements And Main Results: After adjusting for covariates, we found that maternal smoking prior to preterm birth increased the odds of having an infant with BPD by twofold (P = 0.02). Maternal smoking was associated with prolonged mechanical ventilation and respiratory support during the neonatal intensive care unit admission. Preexisting hypertension was associated with a twofold (P = 0.04) increase in odds for BPD. Lower gestational age and birth weight z-scores were associated with BPD. Preterm infants who were exposed to maternal smoking had higher rates of late respiratory disease during childhood. Twenty-two percent of infants diagnosed with BPD and 34% of preterm infants without BPD had no clinical signs of late respiratory disease during early childhood.

Conclusions: We conclude that maternal smoking and hypertension increase the odds for developing BPD after preterm birth, and that maternal smoking is strongly associated with increased odds for late respiratory morbidities during early childhood. These findings suggest that in addition to the BPD diagnosis at 36 weeks, other factors modulate late respiratory outcomes during childhood. We speculate that measures to reduce maternal smoking not only will lower the risk for preterm birth but also will improve late respiratory morbidities after preterm birth.
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http://dx.doi.org/10.1164/rccm.201612-2414OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549867PMC
August 2017

Neurofibromin is a novel regulator of Ras-induced reactive oxygen species production in mice and humans.

Free Radic Biol Med 2016 08 3;97:212-222. Epub 2016 Jun 3.

Department of Pediatrics and Neonatal-Perinatal Medicine, Augusta University, Augusta, GA 30912, United States; Vascular Biology Center, Augusta University, Augusta, GA 30912, United States. Electronic address:

Neurofibromatosis type 1 (NF1) predisposes individuals to early and debilitating cardiovascular disease. Loss of function mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin, leads to accelerated p21(Ras) activity and phosphorylation of multiple downstream kinases, including Erk and Akt. Nf1 heterozygous (Nf1(+/-)) mice develop a robust neointima that mimics human disease. Monocytes/macrophages play a central role in NF1 arterial stenosis as Nf1 mutations in myeloid cells alone are sufficient to reproduce the enhanced neointima observed in Nf1(+/-) mice. Though the molecular mechanisms underlying NF1 arterial stenosis remain elusive, macrophages are important producers of reactive oxygen species (ROS) and Ras activity directly regulates ROS production. Here, we use compound mutant and lineage-restricted mice to demonstrate that Nf1(+/-) macrophages produce excessive ROS, which enhance Nf1(+/-) smooth muscle cell proliferation in vitro and in vivo. Further, use of a specific NADPH oxidase-2 inhibitor to limit ROS production prevents neointima formation in Nf1(+/-) mice. Finally, mononuclear cells from asymptomatic NF1 patients have increased oxidative DNA damage, an indicator of chronic exposure to oxidative stress. These data provide genetic and pharmacologic evidence that excessive exposure to oxidant species underlie NF1 arterial stenosis and provide a platform for designing novels therapies and interventions.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765860PMC
August 2016

Nf1+/- monocytes/macrophages induce neointima formation via CCR2 activation.

Hum Mol Genet 2016 Mar 5;25(6):1129-39. Epub 2016 Jan 5.

Department of Pediatrics and Neonatal-Perinatal Medicine and Vascular Biology Center, Augusta University, Augusta, GA 30912, USA

Persons with neurofibromatosis type 1 (NF1) have a predisposition for premature and severe arterial stenosis. Mutations in the NF1 gene result in decreased expression of neurofibromin, a negative regulator of p21(Ras), and increases Ras signaling. Heterozygous Nf1 (Nf1(+/-)) mice develop a marked arterial stenosis characterized by proliferating smooth muscle cells (SMCs) and a predominance of infiltrating macrophages, which closely resembles arterial lesions from NF1 patients. Interestingly, lineage-restricted inactivation of a single Nf1 allele in monocytes/macrophages is sufficient to recapitulate the phenotype observed in Nf1(+/-) mice and to mobilize proinflammatory CCR2+ monocytes into the peripheral blood. Therefore, we hypothesized that CCR2 receptor activation by its primary ligand monocyte chemotactic protein-1 (MCP-1) is critical for monocyte infiltration into the arterial wall and neointima formation in Nf1(+/-) mice. MCP-1 induces a dose-responsive increase in Nf1(+/-) macrophage migration and proliferation that corresponds with activation of multiple Ras kinases. In addition, Nf1(+/-) SMCs, which express CCR2, demonstrate an enhanced proliferative response to MCP-1 when compared with WT SMCs. To interrogate the role of CCR2 activation on Nf1(+/-) neointima formation, we induced neointima formation by carotid artery ligation in Nf1(+/-) and WT mice with genetic deletion of either MCP1 or CCR2. Loss of MCP-1 or CCR2 expression effectively inhibited Nf1(+/-) neointima formation and reduced macrophage content in the arterial wall. Finally, administration of a CCR2 antagonist significantly reduced Nf1(+/-) neointima formation. These studies identify MCP-1 as a potent chemokine for Nf1(+/-) monocytes/macrophages and CCR2 as a viable therapeutic target for NF1 arterial stenosis.
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http://dx.doi.org/10.1093/hmg/ddv635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764194PMC
March 2016

Preclinical Evidence for the Use of Sunitinib Malate in the Treatment of Plexiform Neurofibromas.

Pediatr Blood Cancer 2016 Feb 16;63(2):206-13. Epub 2015 Sep 16.

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana.

Purpose: Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1(flox/-) pNF murine model.

Experimental Design: Proliferation, β-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1(+/-) mast cells and fibroblasts, respectively. Krox20;Nf1(flox/-) mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [(18)F]DG-PET/CT imaging.

Results: Sunitinib suppressed multiple in vitro gain-in-functions of Nf1(+/-) mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1(flox/-) mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF.

Conclusions: Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1(flox/-) mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials.
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http://dx.doi.org/10.1002/pbc.25763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862309PMC
February 2016

Impaired compensation to femoral artery ligation in diet-induced obese mice is primarily mediated via suppression of collateral growth by Nox2 and p47phox.

Am J Physiol Heart Circ Physiol 2015 Oct 21;309(7):H1207-17. Epub 2015 Aug 21.

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana; Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana;

The present study was undertaken to establish the role of NADPH oxidase (Nox) in impaired vascular compensation to arterial occlusion that occurs in the presence of risk factors associated with oxidative stress. Diet-induced obese (DIO) mice characterized by multiple comorbidities including diabetes and hyperlipidemia were used as a preclinical model. Arterial occlusion was induced by distal femoral artery ligation in lean and DIO mice. Proximal collateral arteries were identified as the site of major (∼70%) vascular resistance to calf perfusion by distal arterial pressures, which decreased from ∼80 to ∼30 mmHg with ligation in both lean and DIO mice. Two weeks after ligation, significant vascular compensation occurred in lean but not DIO mice as evidenced by increased perfusion (147 ± 48% vs. 49 ± 29%) and collateral diameter (151 ± 30% vs. 44 ± 17%). Vascular mRNA expression of p22(phox), Nox2, Nox4, and p47(phox) were all increased in DIO mice. Treatment of DIO mice with either apocynin or Nox2ds-tat or with whole body ablation of either Nox2 or p47(phox) ameliorated the impairment in both collateral growth and hindlimb perfusion. Multiparametric flow cytometry analysis demonstrated elevated levels of circulating monocytes in DIO mice without impaired mobilization and demargination after femoral artery ligation. These results establish collateral resistance as the major limitation to calf perfusion in this preclinical model, demonstrate than monocyte mobilization and demarginatin is not suppressed, implicate Nox2-p47(phox) interactions in the impairment of vascular compensation to arterial occlusion in DIO mice, and suggest that selective Nox component suppression/inhibition may be effective as either primary or adjuvant therapy for claudicants.
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http://dx.doi.org/10.1152/ajpheart.00180.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666923PMC
October 2015

Maturational Changes in Diastolic Longitudinal Myocardial Velocity in Preterm Infants.

J Am Soc Echocardiogr 2015 Sep 27;28(9):1045-52. Epub 2015 May 27.

Section of Critical Care, Pediatric Heart Lung Center, Department of Pediatrics, Children's Hospital Colorado, University of Colorado Denver, Anschutz School of Medicine, Aurora, Colorado.

Background: Doppler tissue imaging (DTI) has been used to evaluate myocardial velocity during ventricular filling, a means of characterizing diastolic function. Previous studies in older children have shown age-related increases in early diastolic tissue velocities, but there are limited data in preterm infants. The aim of this study was to prospectively determine maturational changes in diastolic tissue velocities at two points in time: (1) 7 days of age and (2) 36 weeks' postmenstrual age (PMA). It was further determined whether DTI measures were altered in infants who developed bronchopulmonary dysplasia with or without pulmonary hypertension.

Methods: A total of 277 preterm infants born at <34 weeks' PMA, with birth weights between 500 and 1,250 g, were prospectively enrolled. Echocardiograms were obtained at 7 days of age and repeated at 36 weeks' PMA. Measurements included DTI assessment of early (E') and late (A') annular velocities of the left ventricular free wall, septum and the right ventricular free wall. Statistical analysis included the Wilcoxon rank sum test, simple linear regression, and the χ(2) test.

Results: At 7 days of age, there was a statistically significant increase in the E'/A' ratio as a function of gestational age at birth. At 36 weeks' PMA, E'/A' ratio was increased, but there was no association with gestational age. DTI measures were not different between infants who did or did not develop bronchopulmonary dysplasia or pulmonary hypertension at either time point.

Conclusions: A gestational age-related increase was found in the early diastolic tissue velocities of preterm infants. At a gestational age equivalent to near term, no difference was observed in diastolic tissue velocities, regardless of gestational age at birth. These findings suggest that maturational changes in diastolic function occur relatively independently of the timing of birth.
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http://dx.doi.org/10.1016/j.echo.2015.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567446PMC
September 2015

Clinical significance of monocyte heterogeneity.

Clin Transl Med 2015 14;4. Epub 2015 Feb 14.

Herman B. Wells Center for Pediatric Research, Georgia Regents University, Augusta, Georgia ; Department of Pediatrics and Neonatal-Perinatal Medicine, Indiana University School of Medicine, Indianapolis, Indiana USA ; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 699 Riley Hospital Drive, RR208, Indianapolis, IN 46202 USA.

Monocytes are primitive hematopoietic cells that primarily arise from the bone marrow, circulate in the peripheral blood and give rise to differentiated macrophages. Over the past two decades, considerable attention to monocyte diversity and macrophage polarization has provided contextual clues into the role of myelomonocytic derivatives in human disease. Until recently, human monocytes were subdivided based on expression of the surface marker CD16. "Classical" monocytes express surface markers denoted as CD14(++)CD16(-) and account for greater than 70% of total monocyte count, while "non-classical" monocytes express the CD16 antigen with low CD14 expression (CD14(+)CD16(++)). However, recognition of an intermediate population identified as CD14(++)CD16(+) supports the new paradigm that monocytes are a true heterogeneous population and careful identification of specific subpopulations is necessary for understanding monocyte function in human disease. Comparative studies of monocytes in mice have yielded more dichotomous results based on expression of the Ly6C antigen. In this review, we will discuss the use of monocyte subpopulations as biomarkers of human disease and summarize correlative studies in mice that may yield significant insight into the contribution of each subset to disease pathogenesis.
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http://dx.doi.org/10.1186/s40169-014-0040-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384980PMC
April 2015

Brief report: Endothelial colony-forming cells and inflammatory monocytes in HIV.

J Acquir Immune Defic Syndr 2015 Apr;68(5):550-3

Departments of *Medicine; †Pediatrics, Indiana University School of Medicine, Indianapolis, IN; and ‡Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN (Case is now with Scripps Clinic Medical Group, Scripps Center for Organ & Cell Transplantation, La Jolla, CA).

The relationships between HIV infection, monocyte activation, and endothelial colony-forming cells (ECFCs) are unknown. We compared ECFC, intermediate monocytes (CD14 CD16), and nonclassical monocytes (CD14 CD16) levels in HIV-infected participants virologically suppressed on antiretroviral therapy, HIV-infected treatment-naive participants, and HIV-uninfected healthy controls. ECFC levels were significantly higher in the HIV-infected virologically suppressed group compared with the uninfected controls. CD14 CD16 percentages (but not CD14 CD16 cells) were significantly higher in both HIV-infected groups vs. uninfected controls. In the HIV-infected groups, ECFCs and CD14 CD16 intermediate monocytes were significantly and inversely correlated. Lower availability of ECFCs may partly explain the relationship between greater intermediate monocytes and atherosclerosis in HIV.
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http://dx.doi.org/10.1097/QAI.0000000000000506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357537PMC
April 2015

A multidisciplinary approach in neurofibromatosis 1--authors' reply.

Lancet Neurol 2015 Jan;14(1):30-1

Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA. Electronic address:

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http://dx.doi.org/10.1016/S1474-4422(14)70298-4DOI Listing
January 2015

Lung parenchymal development in premature infants without bronchopulmonary dysplasia.

Pediatr Pulmonol 2015 Dec 2;50(12):1313-9. Epub 2014 Dec 2.

James Whitcomb Riley Hospital for Children Department of Pediatrics, Sections of Pulmonology, Indiana University School of Medicine, Indianapolis, Indiana.

Rationale: While infants who are born extremely premature and develop bronchopulmonary dysplasia (BPD) have impaired alveolar development and decreased pulmonary diffusion (DLCO), it remains unclear whether infants born less premature and do not develop BPD, healthy premature (HP), have impaired parenchymal development. In addition, there is increasing evidence that pro-angiogenic cells are important for vascular development; however, there is little information on the relationship of pro-angiogenic cells to lung growth and development in infants.

Objective: and Methods Determine among healthy premature (HP) and fullterm (FT) infants, whether DLCO and alveolar volume (VA) are related to gestational age at birth (GA), respiratory support during the neonatal period (mechanical ventilation [MV], supplemental oxygen [O2], continuous positive airway pressure [CPAP]), and pro-angiogenic circulating hematopoietic stem/progenitor cells (CHSPCs). We measured DLCO, VA, and CHSPCs in infants between 3-33 months corrected-ages; HP (mean GA = 31.7 wks; N = 48,) and FT (mean GA = 39.3 wks; N =88).

Result: DLCO was significantly higher in HP than FT subjects, while there was no difference in VA , after adjusting for body length, gender, and race. DLCO and VA were not associated with GA, MV and O2; however, higher values were associated with higher CHSPCs, as well as treatment with CPAP.

Conclusion: Our findings suggest that in the absence of extreme premature birth, as well as BPD, prematurity per se, does not impair lung parenchymal development.
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http://dx.doi.org/10.1002/ppul.23134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452454PMC
December 2015

Early pulmonary vascular disease in preterm infants at risk for bronchopulmonary dysplasia.

Am J Respir Crit Care Med 2015 Jan;191(1):87-95

1 Section of Critical Care.

Rationale: Pulmonary hypertension (PH) is associated with poor outcomes among preterm infants with bronchopulmonary dysplasia (BPD), but whether early signs of pulmonary vascular disease are associated with the subsequent development of BPD or PH at 36 weeks post-menstrual age (PMA) is unknown.

Objectives: To prospectively evaluate the relationship of early echocardiogram signs of pulmonary vascular disease in preterm infants to the subsequent development of BPD and late PH (at 36 wk PMA).

Methods: Prospectively enrolled preterm infants with birthweights 500-1,250 g underwent echocardiogram evaluations at 7 days of age (early) and 36 weeks PMA (late). Clinical and echocardiographic data were analyzed to identify early risk factors for BPD and late PH.

Measurements And Main Results: A total of 277 preterm infants completed echocardiogram and BPD assessments at 36 weeks PMA. The median gestational age at birth and birthweight of the infants were 27 weeks and 909 g, respectively. Early PH was identified in 42% of infants, and 14% were diagnosed with late PH. Early PH was a risk factor for increased BPD severity (relative risk, 1.12; 95% confidence interval, 1.03-1.23) and late PH (relative risk, 2.85; 95% confidence interval, 1.28-6.33). Infants with late PH had greater duration of oxygen therapy and increased mortality in the first year of life (P < 0.05).

Conclusions: Early pulmonary vascular disease is associated with the development of BPD and with late PH in preterm infants. Echocardiograms at 7 days of age may be a useful tool to identify infants at high risk for BPD and PH.
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http://dx.doi.org/10.1164/rccm.201409-1594OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299632PMC
January 2015

Neurofibromin-deficient myeloid cells are critical mediators of aneurysm formation in vivo.

Circulation 2014 Mar 26;129(11):1213-24. Epub 2013 Dec 26.

Department of Pediatrics (F.L., B.D.D., L.C.S., J.A.M., M.R.D., W.K.B., K.N.S., D.M.H., J.C., D.W.C., S.J.C., B.K.S., D.A.I.), Wells Center for Pediatric Research (F.L., B.D.D., L.C.S., J.A.M., M.R.D., W.K.B., K.N.S., D.M.H., J.C., D.W.C., S.J.C., B.K.S., D.A.I.), Department of Biochemistry and Molecular Biology (B.D.D., D.W.C., S.J.C., D.A.I.), Microbiology and Immunology (M.R.D.), Pharmacology and Toxicology (L.M.K.), and Neurology (C.M.H.), Indiana University School of Medicine, Indianapolis, IN.

Background: Neurofibromatosis type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity in circulating hematopoietic and vascular wall cells, which are critical for maintaining vessel wall homeostasis. NF1 patients have evidence of chronic inflammation resulting in the development of premature cardiovascular disease, including arterial aneurysms, which may manifest as sudden death. However, the molecular pathogenesis of NF1 aneurysm formation is unknown.

Method And Results: With the use of an angiotensin II-induced aneurysm model, we demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) enhanced aneurysm formation with myeloid cell infiltration and increased oxidative stress in the vessel wall. Using lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1(+/-) aneurysm phenotype in vivo. Finally, oral administration of simvastatin or the antioxidant apocynin reduced aneurysm formation in Nf1(+/-) mice.

Conclusion: These data provide genetic and pharmacological evidence that Nf1(+/-) myeloid cells are the cellular triggers for aneurysm formation in a novel model of NF1 vasculopathy and provide a potential therapeutic target.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.113.006320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960343PMC
March 2014

Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

Am J Pathol 2014 Jan 7;184(1):79-85. Epub 2013 Nov 7.

Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana; Department of Pediatrics and Neonatal-Perinatal Medicine, Indiana University School of Medicine, Indianapolis, Indiana; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana. Electronic address:

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.
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http://dx.doi.org/10.1016/j.ajpath.2013.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873499PMC
January 2014

Membrane and capillary components of lung diffusion and pro-angiogenic cells in infants.

Eur Respir J 2014 Feb 16;43(2):497-504. Epub 2013 May 16.

Indiana University Medical Center, Indianapolis, IN.

Angiogenesis is a critical determinant of alveolarisation, which increases alveolar surface area and pulmonary capillary blood volume in infants; however, our understanding of this process is very limited. The purpose of our study was to measure the pulmonary membrane diffusion capacity (DM) and pulmonary capillary blood volume (VC) components of the diffusing capacity of the lung for carbon monoxide (DLCO) in healthy infants and toddlers, and evaluate whether these components were associated with pro-angiogenic circulating haematopoietic stem/progenitor cells (pCHSPCs) early in life. 21 healthy subjects (11 males), 3-25 months of age, were evaluated. DLCO was measured under normoxic and hyperoxic conditions, and DM and VC were calculated. From 1 mL venous blood, pCHSPCs were quantified by multiparametric flow cytometry. DM and VC increased with increasing body length; however, membrane resistance as a fraction of total resistance to pulmonary diffusion remained constant with somatic size. In addition, DLCO and VC, but not DM, increased with an increasing percentage of pCHSPCs. The parallel increase in the membrane and vascular components of pulmonary diffusion is consistent with alveolarisation during this period of rapid lung growth. In addition, the relationship between pCHSPCs and VC suggest that pro-angiogenic cells may contribute to this vascular process.
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http://dx.doi.org/10.1183/09031936.00016713DOI Listing
February 2014

Normal hematopoiesis and neurofibromin-deficient myeloproliferative disease require Erk.

J Clin Invest 2013 Jan 10;123(1):329-34. Epub 2012 Dec 10.

Herman Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA.

Neurofibromatosis type 1 (NF1) predisposes individuals to the development of juvenile myelomonocytic leukemia (JMML), a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene that encodes a Ras-GTPase-activating protein, results in hyperactivity of Raf/Mek/Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with myriad mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2's differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek/Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption did not grossly compromise myelopoiesis, dual Erk1/2 disruption rapidly ablated granulocyte and monocyte production in vivo, diminished progenitor cell number, and prevented HSPC proliferation in vitro. Genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre(+)Nf1(flox/flox)Erk1(-/-)Erk2(flox/flox)) fully protects against the development of MPD. Collectively, we identified a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly selective Mek/Erk inhibitors in a leukemia that is otherwise resistant to traditional therapy.
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http://dx.doi.org/10.1172/JCI66167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533306PMC
January 2013

Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.

Hum Mol Genet 2013 Mar 29;22(5):977-88. Epub 2012 Nov 29.

Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, IN 46202, USA.

Mutations in the NF1 tumor suppressor gene cause Neurofibromatosis type 1 (NF1). Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity. Some NF1 patients develop cardiovascular disease, which represents an underrecognized disease complication and contributes to excess morbidity and mortality. Specifically, NF1 patients develop arterial occlusion resulting in tissue ischemia and sudden death. Murine studies demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) in bone marrow cells enhances neointima formation following arterial injury. Macrophages infiltrate Nf1(+/-) neointimas, and NF1 patients have increased circulating inflammatory monocytes in their peripheral blood. Therefore, we tested the hypothesis that heterozygous inactivation of Nf1 in myeloid cells is sufficient for neointima formation. Specific ablation of a single copy of the Nf1 gene in myeloid cells alone mobilizes a discrete pro-inflammatory murine monocyte population via a cell autonomous and gene-dosage dependent mechanism. Furthermore, lineage-restricted heterozygous inactivation of Nf1 in myeloid cells is sufficient to reproduce the enhanced neointima formation observed in Nf1(+/-) mice when compared with wild-type controls, and homozygous inactivation of Nf1 in myeloid cells amplified the degree of arterial stenosis after arterial injury. Treatment of Nf1(+/-) mice with rosuvastatin, a stain with anti-inflammatory properties, significantly reduced neointima formation when compared with control. These studies identify neurofibromin-deficient myeloid cells as critical cellular effectors of Nf1(+/-) neointima formation and propose a potential therapeutic for NF1 cardiovascular disease.
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http://dx.doi.org/10.1093/hmg/dds502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561913PMC
March 2013

Imatinib mesylate for plexiform neurofibromas in patients with neurofibromatosis type 1: a phase 2 trial.

Lancet Oncol 2012 Dec 23;13(12):1218-24. Epub 2012 Oct 23.

Department of Pediatrics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

Background: Plexiform neurofibromas are slow-growing chemoradiotherapy-resistant tumours arising in patients with neurofibromatosis type 1 (NF1). Currently, there are no viable therapeutic options for patients with plexiform neurofibromas that cannot be surgically removed because of their proximity to vital body structures. We undertook an open-label phase 2 trial to test whether treatment with imatinib mesylate can decrease the volume burden of clinically significant plexiform neurofibromas in patients with NF1.

Methods: Eligible patients had to be aged 3-65 years, and to have NF1 and a clinically significant plexiform neurofibroma. Patients were treated with daily oral imatinib mesylate at 220 mg/m(2) twice a day for children and 400 mg twice a day for adults for 6 months. The primary endpoint was a 20% or more reduction in plexiform size by sequential volumetric MRI imaging. Clinical data were analysed on an intention-to-treat basis; a secondary analysis was also done for those patients able to take imatinib mesylate for 6 months. This trial is registered with ClinicalTrials.gov, number NCT01673009.

Findings: Six of 36 patients (17%, 95% CI 6-33), enrolled on an intention-to-treat basis, had an objective response to imatinib mesylate, with a 20% or more decrease in tumour volume. Of the 23 patients who received imatinib mesylate for at least 6 months, six (26%, 95% CI 10-48) had a 20% or more decrease in volume of one or more plexiform tumours. The most common adverse events were skin rash (five patients) and oedema with weight gain (six). More serious adverse events included reversible grade 3 neutropenia (two), grade 4 hyperglycaemia (one), and grade 4 increases in aminotransferase concentrations (one).

Interpretation: Imatinib mesylate could be used to treat plexiform neurofibromas in patients with NF1. A multi-institutional clinical trial is warranted to confirm these results.

Funding: Novartis Pharmaceuticals, the Indiana University Simon Cancer Centre, and the Indiana University Herman B Wells Center for Pediatric Research.
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http://dx.doi.org/10.1016/S1470-2045(12)70414-XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380388PMC
December 2012

Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.

Arterioscler Thromb Vasc Biol 2012 Apr 26;32(4):1045-53. Epub 2012 Jan 26.

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, 46202, USA.

Objective: We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy.

Methods And Results: Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells was determined by varying expressions of CD34, CD31, and CD146 but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. We also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol with a prior method, we determined that the present protocol identifies circulating endothelial cells, whereas the earlier protocol identified extracellular vesicles.

Conclusions: Two populations of circulating endothelial cells, including the functionally characterized ECFC, are now identifiable in human cord blood and peripheral blood by PFC.
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http://dx.doi.org/10.1161/ATVBAHA.111.244210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306529PMC
April 2012

Changes in the frequency and in vivo vessel-forming ability of rhesus monkey circulating endothelial colony-forming cells across the lifespan (birth to aged).

Pediatr Res 2012 Feb 21;71(2):156-61. Epub 2011 Dec 21.

Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA.

Introduction: We have identified a novel hierarchy of human endothelial colony-forming cells (ECFCs) that are functionally defined by their proliferative and clonogenic potential and in vivo vessel-forming ability. The rhesus monkey provides an excellent model in which to examine the changes in circulating concentrations and functions of ECFCs since this nonhuman primate possesses a long lifespan and has been used extensively to model age-related processes that occur in humans.

Results: Endothelial cells (ECs) derived from rhesus monkey ECFCs share a cell-surface phenotype similar to human cord blood ECFCs, rapidly form capillary-like structures in vitro, and form endothelial-lined vessels in vivo upon implantation in immunodeficient mice in an age-dependent manner. Of interest, although ECFCs from the oldest monkeys formed capillary-like structures in vitro, the cells failed to form inosculating vessels when implanted in vivo and displayed a deficiency in cytoplasmic vacuolation in vitro; a critical first step in vasculogenesis.

Discussion: Utilizing previously established clonogenic assays for defining different subpopulations of human ECFCs, we have shown that a hierarchy of ECFCs, identical to human cells, can be isolated from the peripheral blood of rhesus monkeys, and that the frequency of the circulating cells varies with age. These studies establish the rhesus monkey as an important preclinical model for evaluating the role and function of circulating ECFCs in vascular homeostasis and aging.

Methods: Peripheral blood samples were collected from 40 healthy rhesus monkeys from birth to 24 years of age for ECFC analysis including immunophenotyping, clonogenic assays, and in vivo vessel formation.
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http://dx.doi.org/10.1038/pr.2011.22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358134PMC
February 2012

Resident Endothelial Progenitor Cells From Human Placenta Have Greater Vasculogenic Potential Than Circulating Endothelial Progenitor Cells From Umbilical Cord Blood.

Cell Med 2012 9;2(3):85-96. Epub 2011 Dec 9.

Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA; †Indiana Center for Vascular Biology and Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; **Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.

Endothelial colony-forming cells (ECFCs) isolated from umbilical cord blood (CBECFCs) are highly proliferative and form blood vessels in vivo. The purpose of this investigation was to isolate and characterize a population of resident ECFCs from the chorionic villi of term human placenta and provide a comparative analysis of their proliferative and vasculogenic potential with CBECFCs. ECFCs were isolated from umbilical cord blood and chorionic villi from placentas obtained by caesarean deliveries. Placental ECFCs (PECFCs) expressed CD144, CD31, CD105, and KDR and were negative for CD45 and CD34, consistent with other ECFC phenotypes. PECFCs were capable of 28.6 ± 6.0 population doublings before reaching senescence (vs. 47.4 ± 3.2 for CBECFCs, p < 0.05, n = 4). In single cell assays, 46.5 ± 1.2% underwent at least one division (vs. 51.0 ± 1.8% of CBECFCs, p = 0.07, n = 6), and of those dividing PECFCs, 71.8 ± 0.9% gave rise to colonies of >500 cells (highly proliferative potential clones) over 14 days (vs. 69.4 ± 0.7% of CBECFCs, p = 0.07, n = 9). PECFCs formed 5.2 ± 0.8 vessels/mm(2) in collagen/fibronectin plugs implanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, whereas CBECFCs formed only 1.7 ± 1.0 vessels/mm(2) (p < 0.05, n = 4). This study demonstrates that circulating CBECFCs and resident PECFCs are identical phenotypically and contain equivalent quantities of high proliferative potential clones. However, PECFCs formed significantly more blood vessels in vivo than CBECFCs, indicating that differences in vasculogenic potential between circulating and resident ECFCs exist.
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http://dx.doi.org/10.3727/215517911X617888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776045PMC
March 2016

Human endothelial colony forming cells undergo vasculogenesis within biphasic calcium phosphate bone tissue engineering constructs.

Acta Biomater 2011 Dec 14;7(12):4222-8. Epub 2011 Jul 14.

Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, 1304 West Green Street, Urbana, IL 61801, USA.

An important consideration in bone regeneration is the need for expedited neovascularization within the defect site. Formation of a vascular network is critical for cell viability and normal function leading to tissue regeneration, but spontaneous angiogenesis is too slow to yield sufficient vessel formation. In this pilot study, human umbilical cord blood (hUCB)-derived endothelial colony forming cells (ECFCs) were evaluated for in vivo vasculogenesis in the macropores of biphasic calcium phosphate (BCP)/bone morphogenetic protein-2 (BMP-2) bone tissue engineering constructs. Constructs were implanted on the abdominal wall of NOD/SCID mice for 4 weeks. This study demonstrated in vivo vasculogenesis by human ECFCs within the macropore space of BCP/BMP-2 constructs. The human ECFC-derived vessels anastomosed with the host vasculature and perfused vessels were visible in the very center of the 5mm diameter, 2.5mm tall scaffolds. Additionally, the vessels were evenly distributed throughout the construct. This study suggests that scaffolds containing ECFCs have significant potential for expedited neovascularization in bony defects.
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http://dx.doi.org/10.1016/j.actbio.2011.07.006DOI Listing
December 2011

Differential mechanisms of x-ray-induced cell death in human endothelial progenitor cells isolated from cord blood and adults.

Radiat Res 2011 Aug 10;176(2):208-16. Epub 2011 Jun 10.

Department of Radiation Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells that circulate at low concentration in human umbilical cord and adult peripheral blood and are largely resident in blood vessels. ECFCs not only appear to be critical for normal vascular homeostasis and repair but may also contribute to tumor angiogenesis and response to therapy. To begin to characterize the potential role of ECFCs during the treatment of tumors in children and adults with radiation, we characterized the X-ray sensitivity of cord and adult blood-derived ECFCs. We found both cord blood and adult ECFCs to be highly radiation sensitive (3 Gy resulted in >90% killing without induction of apoptosis). The X-ray survival curves suggested reduced potential for repair capacity, but X-ray fractionation studies demonstrated that all the ECFCs exhibited repair when the radiation was fractionated. Finally, the mechanisms of X-ray-induced cell death for cord blood and adult ECFCs were different at low and high dose. At low dose, all ECFCs appear to die by mitotic death/catastrophe. However, at high radiation doses (≥ 10 Gy) cord blood ECFCs underwent p53 stabilization and Bax-dependent apoptosis as well as p21-dependent G₁ and G₂/M cell cycle checkpoints. By contrast, after 10 Gy adult ECFCs undergo only large-scale radiation-induced senescence, which is a cellular phenotype linked to premature development of atherosclerosis and vasculopathies. These data demonstrate that the ECFC response to radiation is dose-dependent and developmentally regulated and may provide potential mechanistic insight into their role in tumor and normal tissue response after ionizing radiation treatment.
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http://dx.doi.org/10.1667/rr2427.1DOI Listing
August 2011

Polychromatic flow cytometry identifies novel subsets of circulating cells with angiogenic potential in pediatric solid tumors.

Cytometry B Clin Cytom 2011 Sep 12;80(5):335-8. Epub 2011 May 12.

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA.

Background: Pediatric solid tumors depend upon angiogenesis for their growth and metastases. A new polychromatic flow cytometry (PFC) protocol has revealed circulating cells of hematopoietic and endothelial lineages from the peripheral blood (PB) of healthy individuals, and has defined the different cell types involved in the growth of tumor vasculature that are critical in angiogenesis.

Methods: PB was collected from both healthy children and children with different malignant solid tumors and the mononuclear cells (MNCs) were subsequently isolated. PFC was applied and the MNCs were evaluated for proangiogenic and nonangiogenic circulating progenitor cells (CPCs), endothelial colony forming cells (ECFCs), and mature endothelial cells using the markers CD45, CD31, AC133, CD34, CD14, CD235a, CD41a, and a viability marker.

Results: ECFCs and CPCs were significantly elevated in patients at day 21 compared to controls. The ratio of proangiogenic to nonangiogenic CPCs was significantly elevated compared to controls at baseline and returned to healthy baseline levels following treatment.

Conclusions: We describe the successful identification of these hematopoietic and endothelial progenitor cells in both healthy children and children with solid tumors. In addition, this is a potential discovery of novel predictive biomarkers for future clinical trials.
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http://dx.doi.org/10.1002/cyto.b.20602DOI Listing
September 2011

Gestational diabetes mellitus alters maternal and neonatal circulating endothelial progenitor cell subsets.

Am J Obstet Gynecol 2011 Mar 16;204(3):254.e8-254.e15. Epub 2010 Dec 16.

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

Objective: The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity.

Study Design: Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit-endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging.

Results: Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks' gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit-endothelial cells or colony-forming unit-endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects.

Conclusion: The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities.
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http://dx.doi.org/10.1016/j.ajog.2010.10.913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057499PMC
March 2011

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential.

Cytometry A 2010 Sep;77(9):831-9

Department of Pediatrics, Indiana University, Indianapolis, Indiana, USA.

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34(+)CD45(dim) cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133(+) subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133(+) proangiogenic CHSPCs to AC133(-) nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.
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http://dx.doi.org/10.1002/cyto.a.20921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931367PMC
September 2010

Endothelial abnormalities in adolescents with type 1 diabetes: a biomarker for vascular sequelae?

J Pediatr 2010 Oct 12;157(4):540-6. Epub 2010 Jun 12.

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202-5225, USA.

Objective: To evaluate whether counts of circulating colony forming unit-endothelial cells (CFU-ECs), cells co-expressing CD34, CD133, and CD31 (CD34+CD133+CD31+), and CD34+CD45- cells are altered in adolescents with type 1 diabetes and if the changes in counts correlate with endothelial dysfunction.

Study Design: Adolescents with diabetes (ages 18 to 22 years) and race- and sex-matched control subjects were studied. We assessed circulating CFU-ECs, using colony assays, and CD34+CD133+CD31+ and CD34+CD45- cells, using poly-chromatic flow cytometry. CFU-ECs and CD34+CD133+CD31+ are hematopoietic-derived progenitors that inversely correlate with cardiovascular risk in adults. CD34+CD45- cells are enriched for endothelial cells with robust vasculogenic potential. Vascular reactivity was tested by laser Doppler iontophoresis.

Results: Subjects with diabetes had lower CD34+CD133+CD31+ cells, a trend toward reduced CFU-ECs, and increased CD34+CD45- cells compared with control subjects. Endothelium-dependent vasodilation was impaired in subjects with diabetes, which correlated with reductions in circulating CD34+CD133+CD31+ cells.

Conclusions: Long-term sequelae of type 1 diabetes include vasculopathies. Endothelial progenitor cells promote vascular health by facilitating endothelial integrity and function. Lower CD34+CD133+CD31+ cells may be a harbinger of future macrovascular disease risk. Higher circulating CD34+CD45- cells may reflect ongoing endothelial damage. These cells are potential biomarkers to guide therapeutic interventions to enhance endothelial function and to prevent progression to overt vascular disease.
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http://dx.doi.org/10.1016/j.jpeds.2010.04.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140170PMC
October 2010

Identification of endothelial cells and progenitor cell subsets in human peripheral blood.

Curr Protoc Cytom 2010 Apr;Chapter 9:Unit 9.33.1-11

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA.

An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function. Herein, a reproducible PFC protocol is provided to identify a rare circulating endothelial colony-forming cell (ECFC) with proliferative potential, along with a population of circulating progenitor cells (CPCs) in which the ratio analysis distinguishes between healthy and disease populations. In sum, a reliable PFC protocol, which can be used to investigate the roles of human hematopoietic and endothelial elements in the growth and maintenance of the vasculature, is described.
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http://dx.doi.org/10.1002/0471142956.cy0933s52DOI Listing
April 2010
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