Outgrowth Cells Research Articles

Impairment and Differential Expression of PR3 and MPO on Peripheral Myelomonocytic Cells with Endothelial Properties in Granulomatosis with Polyangiitis

Background. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune-mediated diseases characterized by vasculitic inflammation of respiratory tract and kidneys. Clinical observations indicated a strong association between disease activity and serum levels of certain types of autoantibodies (antineutrophil cytoplasm antibodies with cytoplasmic [cANCA in GPA] or perinuclear [pAN CA in MPA] immunofluorescence). Pathologically, both diseases are characterized by severe microvascular endothelial cell damage. Early endothelial outgrowth cells (eEOCs) have been shown to be critically involved in neovascularization under both physiological and pathological condition. Objectives. The principal aims of our study were (i) to analyze the regenerative activity of the eEOC system and (ii) to determine mPR3 and MPO expression in myelo monocytic cells with endothelial characteristics in GPA and MPA patients. Methods. In 27 GPA and 10 MPA patients, regenerative activity blood-derived eEOCs were analyzed using a culture-forming assay. Flk-1+, CD133+/Flk-1+, mPR3+, and Flk-1+/mPR3+ myelomonocytic cells were quantified by FACS analysis. Serum levels of Angiopoietin-1 and TNF-α were measured by ELISA. Results. We found reduced eEOC regeneration, accompanied by lower serum levels of Angiopoietin-1 in GPA patients as compared to healthy controls. In addition, the total numbers of Flk-1+ myelomonocytic cells in the peripheral circulation were decreased. Membrane PR3 expression was significantly higher in total as well as in Flk-1+ myelomonocytic cells. Expression of MPO was not different between the groups. Conclusions. These data suggest impairment of the eEOC system and a possible role for PR3 in this process in patients suffering from GPA.

Ischemia/Reperfusion Injury, Its Mechanisms, and Prevention

Since the beginning of transplantation era, ischemia/reperfusion injury (IRI) has remained one of the most serious pitfalls of the procedure. Although thousands of scientific studies and analyses have been published, and mechanisms of the injury recognized and described, the most serious step forward to avoid the IRI was Geoffrey Collins' and Folkert Belzer's invention of two different intracellular-type solutions in 1960s, which allowed for safe renal preservation exceeding 24 hours. Since then, no such important improvement took place and prevention of IRI remains a holy grail of transplantation, especially in thoracic and sub-optimal abdominal organs (fatty livers, older donor kidneys). In this issue we present two studies examining various modalities to reduce both cold and warm ischemia. W. Li et al. showed significant improvement in posttransplantation liver function tests, histology, and reduction of apoptosis with administration of sodium nitrite either to preservation solution or directly to the recipient animal. What is more important is that protection was more evident with more pronounced injury, when liver cold ischemia time was extended to 18–24 hours. Although NO is believed to render protection only in specific concentrations, the authors have shown a dose-dependent effect in a 25–250 uM concentration range. In this edition also, M. G. W. van den Heuvel et al. present use of statins in a human model of approximately one hour warm ischemia and reperfusion during breast reconstruction with free cutaneous flap. Although numerous authors postulate usefulness of statins in attenuation of ischemia/reperfusion injury (IRI) [1, 2], employing dozens of pathways and mechanisms in rendering protection, in this study statins did not show any benefit and rather deteriorated the results and increased risk of complications in skin flap warm ischemia and transposition. Some agents can be administered in a gaseous form and A. Siriussawakul et al. give a nice overview of potential applications and proposed mechanisms of action of inhaled nitric oxide, carbon monoxide, and hydrogen sulfide in prevention of IRI and inflammation. Liver is known to tolerate 12 hours of cold ischemia relatively well. However, exceeding preservation beyond this time leads to significant deterioration of both short and long term results of transplantation. This decline is mostly due to biliary pathology, as biliary tree is the most ischemia susceptible tissue of the liver. R. Cursio and J. Gugenheim give a nice overview of biliary complications after liver transplantation. They extensively discuss pathomechanism of ischemic type biliary lesions and the role of cold and warm ischemia as well as intrahepatic cholestasis, bile salts, and immune-dependent events in development of this phenomenon. Posttransplant acute renal failure is mainly caused by ischemia and reperfusion. Although primary damage occurs mainly to tubular cells, microvasculature is also affected, especially with prolonged warm ischemia. This becomes clinically more sound with more and more organs retrieved after cardiac death. Patschan et al. discuss features and mechanism of endothelial cells' injury in renal IRI and potential therapeutic application of endothelial progenitor cells. With the focus of contemporary medicine on regenerative technologies this is definitely a “sexy” topic in IRI, regardless of wether peripheral blood-retrieved endothelial outgrowth cells are true progenitor cells or not. What is more important, several authors mention antifibrogenic properties of such therapy, making it even more attractive in the field of renal transplantation. A key player involved in inflammation, cellular apoptosis and survival in a transplanted organ is a pathway of mitogen activated protein kinases (MAPKs). Despite its essential role in recovery from ischemia, and some new inhibiting agents with potential therapeutic application in the pipeline, this pathway is rather poorly recognized by transplant physicians. We decided to publish a review by G. Vassalli et al., who give a concise description of MAPK pathway in ischemia/reperfusion injury and ischemic preconditioning of the heart. Bases of this knowledge are critical not only to surgeons involved in heart or other organ transplantation, but equally to any other physician treating patients for coronary disease or myocardial infarction. Maciej Kosieradzki Johann Pratschke Jerzy Kupiec-Weglinski Wojciech Rowinski

La depleción de células CD34+/CD144+ en pacientes de revascularización miocárdica no está mediada por un aumento de la apoptosis inducida por el TGF-beta plasmático

IntroductionCoronary atherosclerotic disease has been linked to endothelial progenitor cell (EPC) depletion and functional impairment. We assessed the number of CD34+/KDR+ and CD34+/CD144+ cells in coronary artery bypass grafting (CABG) patients, compared to valvular patients, and the apoptotic effect of the plasma from these two groups on early outgrowth cells (EOCs) from healthy donors. MethodsCD34+/KDR+ and CD34+/CD144+ cell numbers were assessed by flow cytometry. EOCs were obtained from buffy coats from healthy donors. Apoptosis was measured as DNA fragmentation. In vitro expression of CD34 and CD144 was assessed by Western blot. ResultsWe found a lower number of CD34+/CD144+ cells in CABG patients compared to valvular, but a higher number compared to healthy controls. Plasma from CABG patients decreased apoptosis in cultured EOCs from healthy donors (day 7); this effect was abrogated by the TGF-beta1 blockers SIS3 and SB-431542. Prior to apoptosis protection, in cultured cells from healthy donors (day 4), plasma from CABG increased CD34 and CD144 expression, contrary to what found in the blood of the patients. ConclusionsCABG patients possess a lower number of CD34+/CD144+ cells than valvular ones. Plasma from CABG patients is antiapoptotic for EOCs from healthy donors and increases the expression of both CD34 and CD144. Suboptimal number of CD34+/CD144+ in vivo may be mediated by a primary bone marrow defect rather than a deleterious effect of plasma.

Inhibitory Effects of Nisin Against Clostridium perfringens Food Poisoning and Nonfood‐Borne Isolates

The enterotoxigenic Clostridium perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly resistant endospores, it has become a great concern to the meat industry to produce meat free of C. perfringens. In this study, we evaluated the antimicrobial effect of nisin against C. perfringens FP and NFB isolates. No inhibitory effect of nisin was observed against germination of spores of both FP and NFB isolates in laboratory medium. However, nisin effectively arrested outgrowth of germinated spores of C. perfringens in rich medium. Interestingly, germinated spores of NFB isolates possessed higher resistance to nisin than that of FP isolates. Furthermore, nisin exhibited inhibitory effect against vegetative growth of both FP and NFB isolates in laboratory medium, with vegetative cells of NFB isolates showing higher resistance than that of FP isolates. However, the antimicrobial activity of nisin against C. perfringens was significantly decreased in a meat model system. In conclusion, although nisin showed inhibitory effect against spore outgrowth and vegetative cells of C. perfringens FP and NFB isolates in laboratory conditions, no such effect was observed against C. perfringens spores inoculated into a meat model system.

Calcium signalling in adult endothelial outgrowth cells

Endothelial outgrowth cells (EOCs) derived from blood mononuclear cells can differentiate to an endothelial-like phenotype. There are deficits in understanding of the biology of these cells, particularly detailed characterisation of their Ca2+ signalling mechanisms. In the current study, it was found that human EOCs express two forms of ryanodine receptor (RyR1 and RyR2) Ca2+ release channel in their endoplasmic reticulum. Individual EOCs display heterogeneous Ca2+ responses to physiologically relevant regulators fibrinogen and collagen. Some EOCs showed distinctive, multiphasic Ca2+ responses to fibrinogen consisting of rapid decreases, transient increases then a gradual return to the resting levels. Transient elevations in Ca2+ required both L-type voltage gated calcium channels and RyRs. Decreases in Ca2+ stimulated by fibrinogen depended on plasma membrane Ca2+ ATPase pumps, but did not require thapsigargin-sensitive Ca2+ ATPases. These results indicate that EOCs possess sophisticated Ca2+ signalling mechanisms, capable of generating distinct Ca2+ waveforms in response to different physiologically relevant cues.

Double-Edged Role of the CXCL12/CXCR4 Axis in Experimental Myocardial Infarction

Here we assess the intrinsic functions of the chemokine receptor CXCR4 in remodeling after myocardial infarction (MI) using Cxcr4 heterozygous (Cxcr4(+/-)) mice. Myocardial necrosis triggers complex remodeling and inflammatory changes. The chemokine CXCL12 has been implicated in protection and therapeutic regeneration after MI through recruiting angiogenic outgrowth cells, improving neovascularization and cardiac function, but the endogenous role of its receptor CXCR4 is unknown. MI was induced by ligation of the left descending artery. Langendoff perfusion, echocardiography, quantitative immunohistochemistry, flow cytometry, angiogenesis assays, and cardiomyocyte analysis were performed. After 4 weeks, infarct size was reduced in Cxcr4(+/-) mice compared with wild-type mice and in respective bone marrow chimeras compared with controls. This was associated with altered inflammatory cell recruitment, decreased neutrophil content, delayed monocyte infiltration, and a predominance of Gr1(low) over classic Gr1(high) monocytes. Basal coronary flow and its recovery after MI were impaired in Cxcr4(+/-)mice, paralleled by reduced angiogenesis, myocardial vessel density, and endothelial cell count. Notably, no differences in cardiac function were seen in Cxcr4(+/-)mice compared with wild-type mice. Despite defective angiogenesis, Cxcr4(+/-) mouse hearts showed no difference in CXCL12, vascular endothelial growth factor or apoptosis-related gene expression. Electron microscopy revealed lipofuscin-like lipid accumulation in Cxcr4(+/-) mouse hearts and analysis of lipid extracts detected high levels of phosphatidylserine, which protect cardiomyocytes from hypoxic stress in vitro. CXCR4 plays a crucial role in endogenous remodeling processes after MI, contributing to inflammatory/progenitor cell recruitment and neovascularization, whereas its deficiency limits infarct size and causes adaptation to hypoxic stress. This should be carefully scrutinized when devising therapeutic strategies involving the CXCL12/CXCR4 axis.

Fluid Shear Stress Alters the Hemostatic Properties of Endothelial Outgrowth Cells

Surface endothelialization is an attractive means to improve the performance of small diameter vascular grafts. While endothelial outgrowth cells (EOCs) are considered a promising source of autologous endothelium, the ability of EOCs to modulate coagulation-related blood activities is not well understood. The goal of this study was to assess the role of arterial flow conditions on the thrombogenic phenotype of EOCs. EOCs derived from baboon peripheral blood, as well as mature arterial endothelial cells from baboons, were seeded onto adsorbed collagen, then exposed to physiologic levels of fluid shear stress. For important hemostatic pathways, cellular responses to shear stress were characterized at the gene and protein level and confirmed with a functional assay for activated protein C (APC) activity. For EOCs, fluid shear stress upregulated gene and protein expression of anticoagulant and platelet inhibitory factors, including thrombomodulin, tissue factor pathway inhibitor, and nitric oxide synthase 3 (eNOS). Fluid shear stress significantly altered the functional activity of EOCs by increasing APC levels. This study demonstrates that fluid shear stress is an important determinant of EOC hemostatic properties. Accordingly, manipulation of EOC phenotype by mechanical forces may be important for the development of thrombo-resistant surfaces on engineered vascular implants.

Tissue engineering of vascularised human liver organoids: study of morphology phenotypic expression and metabolomics of transitional co-cultures of hepatic/endothelial progenitors

IntroductionTissue architecture and hepatic cell morphology reflect the functional differentiation of the liver. Differentiated functions of hepatocytes, depend on complex, hetereotypic cell-cell/cell-matrix interactions mediated by cell adhesion molecules (CAMs) in...

Suboptimal release of CD34+/CD144+ cells in atherosclerotic patients in response to ischemia: role of plasmatic TGF‐B

ObjectivesTo determine the number of CD34+/CD144+ cells in coronary artery bypass grafting (CABG) patients, compared to valvular patients and healthy controls, and the apoptotic effect of their plasma on peripheral blood‐derived angiogenic early outgrowth cells (EOCs) from healthy donors.MethodsCD34+/CD144+ cell counts were assessed by cell cytometry from peripheral blood mononuclear cells. EOCs were separated by ficcoll gradient and cultured on fibronectin using microvascular medium. Protein expression in EOCs was determined by Western blot.ResultsWe found a lower number of CD34+/CD144+ cells in CABG patients compared to valvular patients, but a higher number compared to controls. Being bypass or valvular had a strong and independent association with CD34+/CD144+ even after the P was adjusted by the number of comparisons. Plasma from CABG patients decreased apoptosis in cultured EOCs from healthy donors (day 7). This effect was abrogated by the transforming growth factor‐beta 1 (TGF‐b1) blockers SIS3 and SB‐431542. Of note, prior to apoptosis protection, plasma from bypass patients was able to upregulate the expression of both CD34 and CD144 at day 4 of isolation.ConclusionBypass patients, which a high degree of ischemia and previous infarct rate, have a suboptimal release of CD34+/CD144+ despite a TGF‐beta‐mediated protective effect of their plasma on cultured EOCs.

Mesenchymal Stem Cell Characteristics of Human Anterior Cruciate Ligament Outgrowth Cells

When ruptured, the anterior cruciate ligament (ACL) of the human knee has limited regenerative potential. However, the goal of this report was to show that the cells that migrate out of the human ACL constitute a rich population of progenitor cells and we hypothesize that they display mesenchymal stem cell (MSC) characteristics when compared with adherent cells derived from bone marrow or collagenase digests from ACL. We show that ACL outgrowth cells are adherent, fibroblastic cells with a surface immunophenotype strongly positive for cluster of differentiation (CD)29, CD44, CD49c, CD73, CD90, CD97, CD105, CD146, and CD166, weakly positive for CD106 and CD14, but negative for CD11c, CD31, CD34, CD40, CD45, CD53, CD74, CD133, CD144, and CD163. Staining for STRO-1 was seen by immunohistochemistry but not flow cytometry. Under suitable culture conditions, the ACL outgrowth-derived MSCs differentiated into chondrocytes, osteoblasts, and adipocytes and showed capacity to self-renew in an in vitro assay of ligamentogenesis. MSCs derived from collagenase digests of ACL tissue and human bone marrow were analyzed in parallel and displayed similar, but not identical, properties. In situ staining of the ACL suggests that the MSCs reside both aligned with the collagenous matrix of the ligament and adjacent to small blood vessels. We conclude that the cells that emigrate from damaged ACLs are MSCs and that they have the potential to provide the basis for a superior, biological repair of this ligament.

Platelet Microparticles Enhance the Vasoregenerative Potential of Angiogenic Early Outgrowth Cells After Vascular Injury

Angiogenic early outgrowth cells (EOCs) have been reported to contribute to endothelial regeneration and to limit neointima formation after vascular injury. Vascular pathologies comprise platelet activation and concomitant generation of platelet microparticles (PMPs). We hypothesized that PMPs may interact with EOCs in the context of vascular injury and modulate their regenerative potential. Using flow cytometry, confocal microscopy, and scanning electron microscopy, we demonstrated the binding of thrombin/collagen-induced PMPs to EOCs with subsequent membrane assimilation and incorporation. This interaction promoted phenotypic alterations of EOCs with increased expression of endothelial cell markers and transfer of the chemokine receptor CXCR4 to EOCs with enhanced responsiveness to its ligand CXCL12/SDF-1alpha. In addition, PMPs augmented the adhesion of EOCs to extracellular matrix components and to the injured vessel wall and accelerated cytoskeletal reorganization and migration of EOCs. PMPs induced changes in the EOC secretome toward a more proangiogenic profile and amplified the EOC-mediated induction of proliferation, migration, and capillary tube formation by mature endothelial cells. Compared with untreated EOCs, the injection of PMP-treated EOCs resulted in accelerated reendothelialization after arterial denudation injury in athymic nude mice, whereas the EOC-mediated reduction of neointima formation remained unchanged. Our data provide evidence that PMPs can boost the potential of EOCs to restore endothelial integrity after vascular injury. Major mechanisms involve the enhancement of EOC recruitment, migration, differentiation, and release of proangiogenic factors.

Granulocyte colony-stimulating factor (G-CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy.

The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G-CSF administration. To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G-CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G-CSF-mobilized samples were collected before administration, at apheresis harvest, and at follow-up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony-forming unit (CFU)-EPC and EOC assays. The effect of exogenous G-CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Our data show that G-CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU-EPCs. Furthermore, G-CSF administration did not mobilize CD34+CD133- cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G-CSF inhibited CFU-EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. G-CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G-CSF may come only from a paracrine effect from HPCs.

Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies

The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar–Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.

Altered adhesive properties of cord blood endothelial outgrowth cells expressing IL‐1ra

The aim of this study was to examine the potential of endothelial outgrowth cells (EOCs) expanded from CD34(+) cord blood-derived cells (CB-EOCs) for overexpression of therapeutic transgenes. As proof of principle, we overexpressed icIL-1ra in CB-EOCs. Proinflammatory activation of CB-EOCs in response to cytokine stimulation (IL-1beta and tumor necrosis factor (TNF)) and during coculture with monocytes showed that icIL-1ra-expressing CB-EOCs express significantly reduced levels of ICAM-1, MCP-1 and thrombin receptor expression. Moreover, overexpression of icIL-1ra attenuated the IL-1beta-mediated proinflammatory activation by diminishing the expression of ICAM-1, SELE, MCP-1 and IL-1beta. Interestingly, overexpression of icIL-1ra also inhibited TNF-induced upregulation of ICAM-1. Expression of ICAM-1, VCAM-1, tissue factor and IL-1beta was also decreased on direct contact with monocytes. These changes in gene expression were accompanied by functional reduction in leukocyte rolling, adhesion of monocytes to CB-EOCs, as well as by a reduction in transendothelial migration of monocytes. Our findings show that CB-EOCs stably expressing transgenic icIL-1ra are protected against activation by not only IL-1beta but also TNFalpha-mediated proinflammatory stimuli and inhibit decisive pathomechanisms of inflammatory processes such as rolling, adhesion and transmigration of monocytes. Therefore, icIL-ra transgenic CB-EOCs may prove to be beneficial in the treatment of IL-1beta- and TNFalpha-mediated inflammatory vasculopathies.

Clinical Summaries

Clinical Summaries

Soluble CD40 Ligand Impairs the Function of Peripheral Blood Angiogenic Outgrowth Cells and Increases Neointimal Formation After Arterial Injury

Recent work has revealed an essential involvement of soluble CD40 ligand (sCD40L) in inflammation and atherosclerosis. We investigated whether sCD40L functionally affects peripheral blood-derived angiogenic early outgrowth cells (EOCs) and neointimal remodeling after arterial injury. Besides myeloid and endothelial markers, cultured human EOCs strongly expressed CD40 mRNA and protein. EOC adhesion to fibronectin, fibrinogen, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 under flow conditions, as well as their transmigration toward stromal cell-derived factor-1alpha, was dose-dependently reduced after preincubation with recombinant human sCD40L for 24 hours. Integrin expression was unaffected by sCD40L, implying that integrin adhesiveness was attenuated. Surface-immobilized CD40L supported much lower adhesion of EOCs than fibronectin. Treatment of EOCs with sCD40L increased superoxide anion production and decreased viability and proliferation. Notably, CD40(-/-) mice displayed reduced neointima and improved re-endothelialization after carotid wire injury compared with wild-type mice, and therapeutic infusion of control EOCs but not EOCs pretreated with sCD40L attenuated neointimal growth after wire injury in nude mice. Furthermore, neointimal growth was more markedly diminished by infusion of spleen-derived CD40(-/-) mouse EOCs than by that of wild-type EOCs. Preincubation of wild-type EOCs but not CD40(-/-) EOCs with sCD40L before their infusion markedly aggravated neointimal formation. Treatment with sCD40L attenuated luminal incorporation of EOCs and accelerated neointimal progression. Endothelial dysfunction due to persistently elevated plasma levels of sCD40L may be attributable to an impairment of EOC function. Hence, in the context of arterial injury, therapeutic blockade of sCD40L may provide a novel strategy for accelerating endothelial regeneration and attenuating neointimal remodeling.

Endothelial Nitric Oxide Synthase as A Marker for Human Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) have been proposed as a promising tool for therapeutic neovascularization, vascular repair, tumor pathology and tissue engineering, though their identification is still a subject of much discussion. EPCs consist of two different subpopulations, termed endothelial cell (EC)-like cells and endothelial outgrowth cells (EOCs). Both types of EPCs are derived from mononuclear cells, but they have different characteristics. Our aim was to characterize and compare the two types of EPCs to find reliable biological features of EPCs that can be used for identification of EPCs. In this study, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and cultured on fibronectin-coated culture plates. While adherent cells were maintained, EC-like cells appeared within 4-7 days of culture, and EOCs developed after 2-3 weeks of culture. EOCs, which were characterized by high proliferation potential, were able to form capillary tubes on Matrigel, but not EC-like cells, despite the higher concentrations of three angiogenic cytokines, vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8, in the conditioned medium of EC-like cells. In contrast, endothelial nitric oxide synthase (eNOS) was expressed in both types of EPCs, and both cell types could produce nitric oxide (NO), as judged by measuring the total amounts of nitrites and nitrates in culture media. In conclusion, the expression of eNOS and the production of NO could be used as common biological features to identify EPCs. These findings provide new insights into the identification of EPCs.

Different expression patterns of organic anion transporting polypeptides in osteosarcomas, bone metastases and aneurysmal bone cysts

Organic anion transporting polypeptides (OATP) were identified as transmembrane transporters for various endo- and exogenous organic compounds (hormones, prostaglandins, anticancer drugs). OATP expression had been shown in different tissues, but not in bone tumors. Therefore, the expression pattern of all known eleven human OATPs was analyzed by quantitative RT-PCR in 21 human bone tumor specimens (osteosarcomas, bone metastases and benign aneurysmal bone cysts). Transcriptional expression of OATP1A2, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1 and 5A1, but not of OATP1B1, 1B3 and 6A1 was observed in malignant and non-malignant tumor specimens at varying level. Importantly, OATP3A1, 4A1, 2B1 and 1C1 mRNA levels were significantly higher in aneurysmal bone cysts as compared to osteosarcomas. Elevated mRNA levels of OATP2A1, 1A2, and 4C1 in metastases from kidney cancer and of OATP5A1 in prostate cancer suggest that the OATP expression pattern in metastases is comparable to that of the primary tumors. Different to tissue, OATP expression in osteosarcoma cell lines HOS and MG-63, normal human osteoblast outgrowth cells (hOB) and bone marrow stromal cells (BMSC) is limited to OATP3A1 and OATP4A1. High OATP expression levels, particularly in benign bone tumors, suggest an important role of these transporters for providing hormones, their conjugates, prostaglandins and drugs in bone cells. Thereby, they may influence bone resorption and formation.

Response to Letter Regarding Article, “Circulating Endothelial Progenitor Cells in Patients With Eisenmenger Syndrome and Idiopathic Pulmonary Arterial Hypertension”

We thank Dr Asosingh et al for their interest in our study.1 Apparent disparity with their data2 probably reflects, at least in part, differences in defining endothelial progenitor cells (EPCs) and the number of patients studied with idiopathic pulmonary arterial hypertension. Asosingh et al2 measured only CD34+ …

Endothelial progenitor cells: identity defined?

In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ, a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133+ cells or CD45+ haematopoietic cells.