Elizabeth M. Skinner

Late Outgrowth Endothelial Cells Resemble Mature Endothelial Cells and Are Not Derived from Bone Marrow

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte‐colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34+CD133−CD146+ cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real‐time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow‐derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34+CD133−CD146+ mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow‐derived circulating precursor for endothelial cells. STEM CELLS2013;31:338–348

Chronic Activation of Corticotropin-Releasing Factor Type 2 Receptors Reveals a Key Role for 5-HT1A Receptor Responsiveness in Mediating Behavioral and Serotonergic Responses to Stressful Challenge

Background The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation. Methods Transgenic mice overexpressing the highly specific CRFR2 ligand urocortin 3 (UCN3OE) were analyzed for stress-related behaviors and hypothalamic-pituitary-adrenal axis responses. Responses to 5-HT receptor agonist challenge were assessed by local cerebral glucose utilization, while 5-HT and 5-hydroxyindoleacetic acid content were quantified in limbic brain regions. Results Mice overexpressing urocortin 3 exhibited increased stress-related behaviors under basal conditions and impaired retention of spatial memory compared with control mice. Following acute stress, unlike control mice, they exhibited no further increase in these stress-related behaviors and showed an attenuated adrenocorticotropic hormone response. 5-HT and 5-hydroxyindoleacetic acid content of limbic nuclei were differentially regulated by stress in UCN3OE mice as compared with control mice. Responses to 5-HT type 1A receptor challenge were significantly and specifically reduced in UCN3OE mice. The distribution pattern of local cerebral glucose utilization and 5-HT type 1A receptor messenger RNA expression levels suggested this effect was mediated in the raphé nuclei. Conclusions Chronic activation of CRFR2 promotes an anxiety-like state, yet with attenuated behavioral and hypothalamic-pituitary-adrenal axis responses to stress. This is reminiscent of stress-related atypical psychiatric syndromes such as posttraumatic stress disorder, chronic fatigue, and chronic pain states. This new understanding indicates CRFR2 antagonism as a potential novel therapeutic target for such disorders.

Impaired vascular function and repair in patients with premature coronary artery disease

Background Endothelial dysfunction is central to the pathogenesis of coronary artery disease, but the role of local and circulating endothelial progenitor cells in maintaining vascular health is poorly understood. We hypothesised that impaired local and circulating vascular repair mechanisms predispose to endothelial dysfunction and the premature onset of coronary artery disease. Methods and results Patients with premature coronary artery disease (n = 16) and healthy age- and sex-matched controls (n = 16) underwent venous occlusion plethysmography with intra-arterial infusion of acetylcholine and sodium nitroprusside. Numbers of circulating endothelial progenitor cells were directly quantified in whole blood by flow cytometry. Endothelial cells were isolated from the blood vessel wall and from peripheral blood mononuclear cells, and expanded in vitro for phenotypic and functional characterisation and analysis of microRNA expression levels. A dose-dependent increase in forearm blood flow (p < 0.001) was attenuated in response to the endothelial-dependent vasodilator acetylcholine in patients compared with controls (p = 0.03). No differences in the number of circulating endothelial progenitor cells or in the phenotype, function or microRNA expression levels of endothelial outgrowth cells isolated from blood were observed in patients and controls. Conversely, local vessel wall endothelial cells from patients had significant impairments in proliferation, adhesion and migration, and significantly reduced expression levels of microRNAs known to regulate endothelial function (miRs −10 a, −let7b, −126 and −181 b) (p < 0.05 for all). Conclusion Local vessel wall derived endothelial cells, rather than circulating endothelial progenitor cells and their progeny, are impaired in patients with vascular dysfunction and premature coronary artery disease.

240 ISOLATION AND CHARACTERISATION OF CORONARY ARTERY ENDOTHELIAL CELLS FROM PATIENTS WITH ACUTE MYOCARDIAL INFARCTION

Introduction Endothelial dysfunction is one of the earliest pathological features in atherosclerosis. Our understanding of endothelial cell biology is derived mainly from studies of human umbilical vein endothelial cells (HUVECs). However HUVECs are not involved in atherosclerosis and provide only limited insight into the pathogenesis of coronary artery disease. We describe a novel method for the isolation of coronary artery endothelial cells from thrombectomy specimens obtained during the treatment of patients for acute myocardial infarction (MI). Methods Patients presenting with ST-segment elevation MI (n=15) who underwent emergency percutaneous coronary intervention (PCI) and thrombus aspiration were recruited. Thrombus specimens were manually dissected, plated onto collagen-I coated plates and maintained in endothelial cell-specific growth media to encourage coronary endothelial outgrowth (CEO) cells. Growth kinetics were evaluated and phenotype was confirmed by immunocytochemical staining for vonWillebrand factor (vWF). Multiparameter flow cytometric analysis was performed (CD31-FITC, KDR-PE, CD146-PE/Cy7 and CD34-APC/Cy; percent positive expression) and angiogenic potential was assessed using an established assay of tubule formation on a matrigel matrix. Results Outgrowth of coronary artery endothelial cells was observed in 9/15 samples. CEO cells were maintained for a minimum of 38 days in culture (mean days=45±12). Population doubling times of CEO cells were comparable to HUVECs (mean±SD: CEO 2.6±0.5, HUVEC 2.3±0.1; p=0.50 student’s t-test). CEO cells had typical cobblestone morphology and expressed vWF. Surface expression of CD31 and KDR was comparable in both cell types (CD31 mean±SD: CEO 74.9±21.3 vs. HUVEC 89.3±11.4, p=0.13; KDR mean±SD: CEO 46.7±31.7 vs. HUVEC 22.2±15.9, p=0.11) whereas CD146 and CD34 expression was increased in CEO cells (CD146 mean±SD: CEO 96±5.3 vs. HUVEC 73.5±30.7, p<0.0001). Conclusion Viable coronary arterial endothelial cells can be isolated from thrombus extracted during emergency PCI. These cells have a mature endothelial phenotype comparable to HUVECs, but have reduced angiogenic potential suggesting they retain the functional characteristics of in situ endothelium. This novel approach to isolate dysfunctional endothelial cells may have applications in drug screening and future studies may provide additional insight into the cellular and molecular basis of endothelial dysfunction in patients with coronary artery disease.

193 THE IMPORTANCE OF CELL SOURCE FOR SOMATIC TISSUE REPROGRAMMING: ENDOTHELIAL CELL-DERIVED IPS CELLS HAVE ENHANCED CAPACITY TO DIFFERENTIATE INTO FUNCTIONAL ENDOTHELIAL CELLS

Background Induced pluripotent stem (iPS) cell technology has primarily focused on the reprogramming of fibroblasts to an embryonic stem (ES)-like state. However, as the field has developed, the importance of somatic cell source has been studied to enhance reprogramming and differentiation efficiencies. Objectives To generate iPS cells from human fibroblasts and endothelial outgrowth cells from blood. To compare reprogramming efficiency between both cell types, and compare their potential for endothelial differentiation. Methods and Results Reprogramming: Episomal vectors containing Sox2, Klf4, Oct4 and c-Myc were electroporated into fibroblasts and endothelial cells using the Amaxa system. Successfully reprogrammed fibroblast-derived iPS (’fiPS’) and endothelial cell-derived iPS (‘eiPS’) arose as colonies, and were picked and expanded. Reprogrammed cells expressed pluripotency markers SSEA3, SSEA4, TRA-1-60, Oct4 and NANOG, and developed into all three germ layers following embryoid body (EB) formation. Optimisation of endothelial differentiation protocol: iPS and ES cell lines were aggregated into EBs for three days in stem cell growth media containing mesoderm-inducing cytokines. EBs were then disaggregated and cultured in Endothelial Growth Medium supplemented with VEGF. After seven days, a population of CD31+ cells was isolated by FACS sorting, cultured and mature endothelial cell antigen expression determined using flow cytometry. CD31+ cells were selected for functional assessment in vitro using established assays of angiogenesis, migration and adhesion. Human endothelial cells derived from iPS cells were implanted subcutaneously in a NOD-SCID mouse model of angiogenesis and neovasculogenesis quantified at day 21. Comparison of fiPS and eiPS: eiPS differentiate into endothelial cells with greater efficiency than fiPS (CD31+ cells at day seven is 15.2% and 4.1%, respectively). fiPS-endothelial cells have been characterized phenotypically and shown to express endothelial markers CD146 (86.2%±10.1%), CD31 (92.9%±3.3%), VEFGR2 (44.8%±2.8%), Tie-2 (30.7%±19%) and VE-Cadherin (65.0%±7.1%). When grown on Matrigel %, they form tubule-like structures with a similar number of vessel connections per field to control endothelial cells (54.5±5.5 versus 57.5±4.0). Characterisation of eiPS-endothelial cells is ongoing. In vivo, implantation of endothelial cells derived from fiPS and eiPS increase vessel formation by 78.2%±16.4% and 67.2%±7.6% respectively, compared to Matrigel - control. By comparison control endothelial cells increased vessel formation by 6.9%±0.8%. Conclusion Endothelial cells can be isolated from blood and reprogrammed effectively to form eiPS cell lines with greater capacity to differentiate into endothelial cells than iPS cells derived from dermal fibroblasts suggesting reprogrammed cells retain epigenetic memory. Endothelial cells derived from both eiPS and fiPS cells increase angiogenesis compared to mature endothelial cells and have potential therapeutic applications for vascular regeneration.