Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle

Trafficking of transplanted endothelial progenitor cells (EPCs) to ischemic tissue is enhanced by stromal-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF). However, it has not been studied how these cytokines modulate the local milieu to entrap EPCs. This study was performed to elucidate a molecular pathway of trafficking EPCs through Akt and to test its application as an adjuvant modality to increase EPC homing. In a mouse hind limb ischemia model, systemically administered 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled mouse EPCs showed three stages of homing to ischemic limb: adhesion to endothelial cells (ECs), incorporation to capillary, and transendothelial migration into extravascular space. As an underlying mechanism to control adhesion of EPCs to ECs, we found that Akt was activated in ECs of ischemic muscle by ischemia-induced VEGF and SDF-1. In vitro and in vivo experiments using adenoviral vector for constitutively active or dominant-negative Akt genes showed that activated Akt enhanced intercellular adhesion molecule 1 (ICAM-1) expression on ECs. Akt activation in ECs also enhanced EPC incorporation to ECs and transendothelial migration in vitro experiments. Activated Akt was sufficient for induction of EPC homing even in normal hind limb, where VEGF or SDF-1 was not increased. Finally, local Akt gene transfer to ischemic limb significantly enhanced homing of systemically administered EPCs, new vessel formation, blood flow recovery, and tissue healing. Akt plays a key role in EPC homing to ischemic limb by controlling ICAM-1 and transendothelial migration. Modulation of Akt in the target tissue may be an adjunctive measure to enhance homing of systemically administered stem cells, suggesting a possibility of cell-and-gene hybrid therapy. Disclosure of potential conflicts of interest is found at the end of this article.     

内皮祖细胞在血管组织工程的应用

内皮祖细胞是一群具有游走特性,能进一步增殖分化成为成熟内皮细胞的幼稚内皮细胞.内皮祖细胞参与了出生后缺血组织的血管发生和血管损伤后的修复.内皮祖细胞的发现为血管组织工程种子细胞增添了一个新来源.本文重点介绍成体内皮祖细胞的来源、鉴定、生物学特性、功能以及在血管组织工程中的应用.     

Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPC-transplanted group exhibited significantly more monocytes/macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization.     

New strategies for in vivo tissue engineering by mimicry of homing factors for self-endothelialisation of blood contacting materials

For years intensive research has been done to endothelialise vascular prostheses with autologous endothelial cells before implantation in patients. However, this procedure is extremely time-, labor- and cost-intensive and can be realized only in very few clinical cases. The discovery of circulating endothelial progenitor cells (EPCs) in 1997 brought new perspectives for the endothelialisation of blood contacting materials. Coating of synthetic graft surfaces with capture molecules for circulating EPCs mimics a pro-homing substrate for fishing out EPCs directly from the bloodstream after implantation. These cells with high proliferation potential can cover the graft with non-thrombogenic endothelium which maintains optimal haemostasis and minimize the risk of restenosis. In this review, different concepts are discussed to capture circulating EPCs on synthetic vascular grafts after implantation. We hypothesize that in vivo self-endothelialisation of blood contacting materials by homing factor-mimetic capture molecules for EPCs may bring revolutionary new perspectives towards future clinical application of stem cell and tissue engineering strategies.     

The Rho kinase inhibitor fasudil augments the number of functional endothelial progenitor cells in ex vivo cultures

Rho kinase (ROCK) has been implicated in the regulation of vascular tone, endothelial dysfunction, inflammation and remodeling. Endothelial progenitor cells (EPC) have been proven to have the efficacy of therapeutic neovascularization in ischemia. However, the scarcity of EPCs limits cell therapy. Using an in vitro EPC culture assay, Y27632 was found to increase the number of adherent EPCs. In this study, we investigated the effect of fasudil, another ROCK inhibitor being used in the clinic, on EPC number and examined whether EPCs expanded by fasudil are functional in vitro and in vivo. In ex vivo cultures of EPCs, fasudil effectively increased the number of ac-LDL/UEA-1 positive cells as well as adherent cells, in contrast to H89, a less selective ROCK inhibitor. Fasudil also increased EPC numbers in culture up to 10 μM, in a dose-dependent manner. When EPCs expanded with fasudil were examined for the migratory activity toward stromal cell-derived factor-1 and vascular endothelial growth factor, these cells retained functional properties in migration, albeit with some decrease. Fasudil-cultured EPCs labeled with PKH26 showed an activity similar to non-treated EPCs for cellular adhesion into an endothelial cell (EC) monolayer and incorporation into capillary-like structures formed by ECs. Finally, when EPCs cultured with fasudil (106 cells/mouse) were injected into ischemic limbs, these cells showed a blood flow recovery at a level comparable to non-treated control EPCs and increased neovascularization. Therefore, these data suggest that the ROCK inhibitor fasudil can provide a beneficial effect in the treatment of ischemic diseases by increasing EPC numbers.     

Potential of baboon endothelial progenitor cells for tissue engineered vascular grafts

Thrombosis and intimal hyperplasia limit the usefulness of small caliber vascular grafts. While some improvements have been reported for grafts seeded with mature endothelial cells (EC), the harvesting of ECs from autologous sources, for example, veins or adipose tissue, remains problematic. More recently, endothelial progenitor cells (EPCs) have been considered a promising source of ECs because EPCs can be readily isolated from whole blood then rapidly expanded in vitro. Additionally, EPCs are increasingly recognized to play important roles in hemostasis, angiogenesis, and arterial injury repair. However, the characterization of EPCs in relevant animal models remains poorly defined. Accordingly, we have characterized the isolation, growth, and functional characteristics of Baboon EPCs (BaEPCs) to evaluate their potential for an autologous cell source for tissue engineered vascular grafts. BaEPCs were successfully cultured from the peripheral blood with an average population doubling time of 1.17 +/- 0.43 days. While the BaEPCs were positive for typical EC markers of vWF, CD31, VE-cadherin, VEGF-R2, Thrombomodulin, and E-selectin, there was reduced eNOS expression. The BaEPCs cell body and actin filaments align in the direction of flow typical of mature ECs. Thus while the lack of eNOS expression is worthy of investigation, EPCs are an attractive cell source for tissue engineered vascular grafts and the baboon model has great potential for continuing evaluations of these cells.     

自体血管内皮祖细胞治疗缺血缺氧性脑损伤

背景：缺血缺氧性脑损伤后的神经再生、神经功能的恢复与缺血部位新生血管的形成和重塑有着密切的关系。血管内皮祖细胞参与出生后缺血组织的血管新生及修复,促使血液循环再通和氧气等营养物质的供应,为神经功能的恢复提供微环境。 目的：探讨自体血管内皮祖细胞治疗缺血缺氧性脑损伤的可行性、有效性及安全性,探索改善脑损伤患者神经功能修复的新方法。 方法：应用计算机检索PubMed、ScienceDirect、Springerlink、CNKI、万方等数据库近10年的相关文献。英文关键词为“EPCs、endothelial progenitor cell、stroke”等,中文关键词为“内皮祖细胞、干细胞移植、脑卒中”等,选择内容与血管内皮祖细胞治疗缺血缺氧性疾病相关的文献,同一领域文献则选近期发表的或发表在权威杂志上的文章,共纳入43篇参考文献。 结果与结论：脑缺血后神经再生、神经功能的恢复与缺血部位新生血管的形成和重塑有着密切的关系,内皮祖细胞参与出生后缺血组织的血管发生及修复,促进血液循环及氧气等营养物质的供应,为神经功能的恢复提供微环境。自体血管内皮祖细胞治疗缺血缺氧性脑损伤是可行的、安全的、有效的,但仍需大量的生物学及动物实验为其临床应用提供客观的理论依据。     

A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31(-);CD146(-) SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.     

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo. Microfluidic micropatterning was used to create 15-microm wide adhesive lanes with 45-microm spacing to reproduce the elongated EC morphology without the influence of external forces. Both EPCs and ECs elongated on micropatterned lanes had aligned actin cytoskeleton and readily deposited ECM. EPCs deposited and remodeled the ECM to a greater extent than ECs. Since a readily produced ECM can improve graft patency, EPCs are an advantageous cell source for endothelializing vascular constructs. Furthermore, EC deposition of ECM was dependent on cell morphology, where elongated ECs deposited more collagen IV and less fibronectin compared to matched cobblestone controls. Thus micropatterned surfaces controlled EC shape and ECM deposition, which ultimately has implications for the design of tissue-engineered vascular constructs.     

Transfection of VEGF(165) genes into endothelial progenitor cells and in vivo imaging using quantum dots in an ischemia hind limb model

Endothelial progenitor cells (EPCs) were transfected with fluorescently labeled quantum dot nanoparticles (QD NPs) with or without VEGF(165) plasmid DNA (pDNA) to probe the EPCs after in?vivo transplantation and to test whether they presented as differentiated endothelial cells (ECs). Bare QD NPs and QD NPs coated with PEI or PEI?+?VEGF(165) genes were characterized by dynamic light scattering, scanning electron microscopy, and atomic force microscopy. Transfection of EPCs with VEGF(165) led to the expression of specific genes and proteins for mature ECs. A hind limb ischemia model was generated in nude mice, and VEGF(165) gene-transfected EPCs were transplanted intramuscularly into the ischemic limbs. At 28 days after transplantation, the VEGF(165) gene-transfected EPCs significantly increased the number of differentiated ECs compared with the injection of medium or bare EPCs without VEGF(165) genes. Laser Doppler imaging revealed that blood perfusion levels were increased significantly by VEGF(165) gene-transfected EPCs compared to EPCs without VEGF(165). Moreover, the transplantation of VEGF(165) gene-transfected EPCs increased the specific gene and protein expression levels of mature EC markers and angiogenic factors in the animal model.     

Isolation of endothelial progenitor cells from cord blood and induction of differentiation by ex vivo expansion

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1 beta , fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.     

内皮祖细胞与肢体缺血疾病的研究现状简

 肢体缺血性疾病已成为发达国家及许多发展中国家的主要疾病之一,而干细胞移植是治疗肢体缺血性疾病备受关注的新方法。内皮祖细胞( EPCs)可分化为内皮细胞,参与新生血管生成。研究证实, 体内移植EPCs可以促进缺血后组织内的血管新生, 为缺血性疾病治疗提供了新策略。     

Enhanced dermal wound neovascularization by targeted delivery of endothelial progenitor cells using an RGD-g-PLLA scaffold

Endothelial progenitor cells (EPCs), endothelial precursors that promote neovascularization in ischemic tissues, have shown the limited vascular regeneration efficacy due to their poor homing into injured sites and low survival, so that a variety of biosynthetic scaffolds have been employed as cell delivery vehicles to overcome the current cell transplantation methods. However, few paralleled studies that directly compare the efficacy of EPCs seeded within synthetic scaffolds to that of EPCs delivered by the conventional transplantation techniques used for EPC therapies have been performed. To address these issues, RGD-g-PLLA biosynthetic scaffold was developed for the targeted EPC delivery and was found to successfully support the in vitro growth and endothelial functions of EPCs. This scaffold also appeared to be good as in vivo targeted delivery carriers of EPCs as it promoted vascular regeneration in a murine dermal wound models. Furthermore, direct comparison with the intradermal EPC injection revealed that the targeted delivery of EPCs by using the RGD-g-PLLA scaffold was superior to their conventional local injection method in terms of the localization and survival/retention of the transplanted EPCs, and their vascular repairing potential. These results suggest that the development of an effective stem cell delivery system may help to maximize the tissue-repairing efficacy with a limited number of stem cells, thereby resolving the limited clinical success of current stem cell therapies that have utilized simple cell injections or infusions.     

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34⁺ EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases.     

Endothelial progenitor cells: a novel tool for the therapy of ischemic diseases

Circulating endothelial progenitor cells (EPCs) are believed to home to sites of neovascularization, contributing to vascular regeneration either directly via incorporation into newly forming vascular structures or indirectly via the secretion of pro-angiogenic growth factors, thereby enhancing the overall vascular and hemodynamic recovery of ischemic tissues. The therapeutic application of EPCs has been shown to be effective in animal models of ischemia, and we as well as other groups involved in clinical trials have demonstrated that the use of EPCs was safe and feasible for the treatment of critical limb ischemia and cardiovascular diseases. However, many issues in the field of EPC biology, especially in regard to the proper and unambiguous molecular characterization of these cells, still remain unresolved, hampering not only basic research but also the effective therapeutic use and widespread application of these cells. Further, recent evidence suggests that several diseases and pathological conditions are correlated with a reduction in the number and biological activity of EPCs, making the development of novel strategies to overcome the current limitations and shortcomings of this promising but still limited therapeutic tool by refinement and improvement of EPC purification, expansion, and administration techniques, a rather pressing issue.     

犬骨髓内皮祖细胞生物学特性及诱导分化

目的:探讨犬骨髓内皮祖细胞(EPCs)随培养时间延长其生物学特性变化及向内皮细胞(ECs)分化能力。方法:免疫微珠分选方法纯化犬骨髓CD14 细胞,EGM-MV2条件培养基培养,于不同时间检测EPCs的生物学特性;将存活至8周的EPCs以分化培养基(M199 20%胎牛血清 VEGF)诱导培养,检测ECs表面标志的表达。结果:早期EPCs为分选后8代之前的EPC,体积略小,类圆形,60%融合时呈串珠样排列,表达CD133、CD14;晚期EPCs由表达VE-cadherin、KDR、CD14的早期EPCs分化而来,形态为多边形,分泌活动明显加强,表达CD133、VE-cadherin、CD14和KDR;诱导分化细胞呈鹅卵石样外观,表达Ⅷ因子、CD31。结论:犬骨髓EPCs随培养时间呈现不同生物学特性,早期EPCs不稳定,晚期EPCs显示出更稳定的生物学特性,能够分化为ECs,是血管组织工程学研究良好的种子细胞。     

Endothelial progenitor cells and cerebrovascular diseases

Identifying factors that may increase the risk of stroke and assessing if treatment of such conditions may lower that risk are important in the management of cerebrovascular disease. Tobacco smoking, poor diet, hypertension and hyperlipidemia remain the major risk factors, and treatment of these conditions has been shown to significantly reduce stroke. In recent years, research has shown that stem cells from a variety of sources can be used as a tool to study and prevent the events that lead to stroke. In this regard, a population of adult stem cells, called endothelial progenitor cells (EPCs), have been identified in peripheral blood and may play an important role in tissue vascularization and endothelium homeostasis in the adult. Most of the studies on EPCs have been carried out on patients with cardiovascular diseases; however, there is emerging evidence which suggests that the introduction or mobilization of EPCs can restore tissue vascularization even after cerebrovascular diseases (CVD), such as ischemic stroke or intracerebral haemorrhage. In this review, we discuss the present level of knowledge about the characteristics of EPCs, their possible therapeutic role in CVD and how they could alter clinical practice in the future.     

Human endothelial progenitor cell-seeded hybrid graft: proliferative and antithrombogenic potentials in vitro and fabrication processing

In this article, we show that human endothelial progenitor cells (EPCs) in circulating peripheral blood may be a novel cell source for a cell-incorporated engineered vascular graft. Cultures of human peripheral blood mononuclear cells collected by the density gradient technique developed highly proliferative EPC colonies, which expanded with culture time. The production rates of antiplatelet substances such as endothelial-type nitric oxide synthase and 6-keto-prostaglandin-F(1)-alpha were approximately one-third and approximately one-half of those of mature endothelial cells (ECs), respectively. On the other hand, the tissue-type plasminogen activator production rate of EPCs was almost the same as that of ECs. EPCs were seeded and cultured on a small-diameter compliant graft (inner diameter, 1.5 mm) made of microporous segmented polyurethane film coated with a photo-reactive gelatin layer, and subsequently subjected to hydrodynamic shear stress by ex vivo circulation. EPCs fully covering the graft elongated and aligned themselves with the direction of the flow, resulting in the production of an integrated EPC-engineered graft. These results indicate that EPCs, which have high proliferative potential and high antithrombogenic potential, comparable to those of ECs, are a suitable cell source for cardiovascular tissue engineering.     

Endothelial progenitor cells and cerebrovascular diseases

Identifying factors that may increase the risk of stroke and assessing if treatment of such conditions may lower that risk are important in the management of cerebrovascular disease. Tobacco smoking, poor diet, hypertension and hyperlipidemia remain the major risk factors, and treatment of these conditions has been shown to significantly reduce stroke. In recent years, research has shown that stem cells from a variety of sources can be used as a tool to study and prevent the events that lead to stroke. In this regard, a population of adult stem cells, called endothelial progenitor cells (EPCs), have been identified in peripheral blood and may play an important role in tissue vascularization and endothelium homeostasis in the adult. Most of the studies on EPCs have been carried out on patients with cardiovascular diseases; however, there is emerging evidence which suggests that the introduction or mobilization of EPCs can restore tissue vascularization even after cerebrovascular diseases (CVD), such as ischemic stroke or intracerebral haemorrhage. In this review, we discuss the present level of knowledge about the characteristics of EPCs, their possible therapeutic role in CVD and how they could alter clinical practice in the future.     

Characterization of porcine circulating progenitor cells: toward a functional endothelium

The lack of available healthy vessels, significant patient morbidity, and high costs hinders the successful clinical utilization of autologous endothelial cells (ECs). Herein we assess the feasibility of using endothelial progenitor cells (EPC) found in circulating blood to engineer a functional endothelium on poly(1,8-octanediol-co-citrate) (POC), a hemocompatible and biodegradable elastomer used in vascular tissue engineering. EPCs were isolated from porcine blood and biochemically differentiated into porcine endothelial (PE)-like cells in vitro. Once differentiated, EC phenotype and function on POC were assessed according to the presence of the EC-specific markers von Willebrand factor, platelet EC adhesion molecule, and vascular endothelial cadherin; metabolism of acetylated low-density lipoprotein; secretion of the anti-thrombogenic factors nitric oxide and prostacyclin; and inhibition of platelet adhesion and clotting processes in vitro. The effects of PE-like cells on porcine aortic smooth muscle cells (PASMCs) were also investigated via co-culture. PE-like cells on POC had phenotype, function, and clotting responses similar to those of primary aortic ECs. The presence of PE-like cells resulted in a 71 +/- 20% decrease in PASMC proliferation; a 52 +/- 2% decrease in the protein:deoxyribonucleic acid ratio; and an elongated, spindle-shaped morphology indicative of a shift from the proliferative to the contractile phenotype. These data suggest that EPCs and POC can provide the basis for a functional tissue-engineered endothelium.