Hematopoietic stem and progenitor cell trafficking

Migration of hematopoietic stem cells (HSCs) is essential during embryonic development and throughout adult life. During embryogenesis, trafficking of HSCs is responsible for the sequential colonization of different hematopoietic organs by blood-producing cells. In adulthood, circulation of HSCs maintains homeostasis of the hematopoietic system and participates in innate immune responses. HSC trafficking is also crucial in clinical settings such as bone marrow (BM) and stem cell transplantation. This review provides an overview of the molecular and cellular signals that control and fine-tune trafficking of HSCs and hematopoietic progenitor cells in embryogenesis and during postnatal life. We also discuss the potential clinical utility of therapeutic approaches to modulate HSC trafficking in patients.     

The use of magnetic resonance cell tracking to monitor endothelial progenitor cells in a rat hindlimb ischemic model

A water-soluble magnetic resonance imaging (MRI) contrast agent, Dextran mono-N-succinimidyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-gadolinium³⁺ (Dex-DOTA-Gd³⁺), was shown to enable monitoring of the anatomical migration and the survival period of transplanted stem cells for up to 1 month. Gadolinium molecules in the cells were rapidly eliminated from the site and excreted upon cell death. Endothelial progenitor cells (EPCs) transplanted into the inguinal femoral muscle of rats migrated distally through the knee in rats after hindlimb ischemia but did not migrate in non-ischemic rats. Interestingly, the survival period of transplanted EPCs was notably prolonged in the ischemic limb, indicating that EPCs are required by the ischemic tissues and that the fate of transplanted EPCs was affected by the disease. Compared to the commonly used particle type of MRI contrast agents, the system described in this study is expected to be invaluable to help clarifying the process of stem cell transplantation therapy.     

Nicotine enlivenment of blood flow recovery following endothelial progenitor cell transplantation into ischemic hindlimb

Nicotine has been demonstrated to stimulate postnatal angiogenesis, having an antiapoptotic effect on endothelial cells. Given the extent of this angiogenesis-promoting effect, we hypothesized that nicotine may also stimulate postnatal vasculogenesis on endothelial progenitor cells (EPCs). Our analyses reveal some intriguing results using an in vitro assay with 2 x 10(-6) M of nicotine (smoker's average nicotine concentration and the dose of nicotine replacement therapy). The proliferation and migration activities of human EPCs cultured from peripheral blood mononuclear cells of non-smoking healthy volunteers were not affected by nicotine. The effect of nicotine on EPC survival was significantly enhanced under serum starvation on the ratio of Hoechest 33342-stained pyknotic nuclear cells as well as Annexin-V-stained cells to total cells. Furthermore, the antiapoptotic effect of nicotine was blocked completely by nicotinic acetylcholine receptor (nAChR) antagonist hexamethonium. Next, we verified how nicotine acts in vivo. Nicotine (100 ng/ml) was administered orally for 7 days before and 4 weeks after injection of cultured EPCs (1 x 10(5) /mouse) into the tail veins of 8-week-old athymic nude mice with ischemic hindlimbs. Laser doppler imaging analysis indicated that blood perfusion in the ischemic hindlimb was significantly enhanced in EPCs plus nicotine, as compared with EPCs alone. These findings suggest nicotine improves blood flow following EPC transplantation in patients with ischemic diseases.     

内皮祖细胞临床应用的研究进展

内皮祖细胞是内皮细胞的前体细胞,在某些生理、病理状态下,可随血液流至相应组织,分化为内皮细胞,并进一步形成血管。内皮祖细胞参与血管新生的作用,对于创伤修复、缺血性疾病的治疗以及肿瘤的靶向治疗具有重要意义,在临床上具有广阔的应用前景。     

P13K／Akt信号传导通路在大鼠骨髓来源内皮祖细胞分化中作用的研究

目的探讨P13K／Akt在内皮祖细胞（EPC）分化中的作用。方法密度梯度离心法分离大鼠骨髓EPC,经差速法接种2次贴壁细胞：采用激光共聚焦显微镜鉴定培养5dEPC;Westemblot检测第O、3、7、10、14天EPC中AC133、vWF、P13K、Akt蛋白表达水平;并在第3、7、10天加入LY294002作用12h,用RT—PCR检测AC133、vWFmRNA水平,Westernb10t检测p—Akt水平。结果经Westemblot检测,AC133在0d表达最强,第3天有弱表达,第7、10、14天几乎无表达（P〈0．05）：vWF表达强度没有明显变化（P〉0．05）;P13K和Akt在0d表达最强,第3天表达稍弱,随培养时间的延长表达逐渐减弱：LY294002作用12h后,第7天和第10天AC133mRNA表达水平低于第3天的水平（P〈0．05）;vWF的表达没有明显变化（P〉0．05）;p—Akt蛋白表达逐渐下降,第10天与第3天相比差异有统计学意义（P〈0．05）。结论EPC分化为内皮细胞过程中．可能有P13K／Akt的参与,     

RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. In this study, we demonstrate a novel and critical role for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models led to apoptosis and failures in tube formation. We identified a novel link between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy revealed that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. We show that this colocalization is functionally relevant, because when RhoB was depleted, Akt was excluded from the nucleus and total cellular Akt protein was decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to only be rate limiting for endothelial cell sprouting, we propose that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable.     

血管内皮祖细胞与缺血性心血管病的血管新生

1997年Ｔａｋａｙｕｋｉ等[1] 在Ｓｃｉｅｎｃｅ杂志发表论文报道血管内皮祖细胞 (ｅｎｄｏｔｈｅｌｉａｌｐｒｏｇｅｎｉｔｏｒｃｅｌｌｓ ,ＥＰＣ)分离成功 ,并可用于体内血管新生 (ａｎｇｉｏｇｅｎｅｓｉｓ)。当时提出的血管内皮祖细胞认为是假定的 (ｐｕｔａｔｉｖｅ)。但是 ,近年来随着干细胞研究的深入 ,血管内皮祖细胞的存在基本已经确认 ,而且它与血管新生间的关系日益受到重视[2 ,3 ] 。本文将讨论血管内皮祖细胞的特征 ,它与缺血性心血管疾病中血管新生的关系及其可能的应用前景。1　血管内皮祖细胞的特征　　血管内皮祖细胞是血…     

内皮祖细胞与缺血性脑血管疾病的治疗

背景：近年来大量基础研究发现,在成年机体的骨髓及外周血中均能找到内皮祖细胞,其在成体病理条件下对血管再生与新生过程起着重要作用。脑梗死发生后,在缺血部位存在着新血管形成,而此过程同样存在着内皮祖细胞的参与,内皮祖细胞的增加以及最大程度参与到新血管组织中,这些很可能是脑血管疾病治疗的一条重要途径。 目的：以文献资料分析法探讨分析内皮祖细胞的特性和作用,及其在缺血性脑血管疾病中的应用进展。 方法：检索SCI数据库、CNKI数据库2002至2011年有关内皮祖细胞与缺血性脑血管疾病治疗的相关文献,检索词为“内皮祖细胞(endothelial progenitor cells, EPCs);前体细胞(precursor cell);脑卒中(stroke);脑缺血(brain ischemia, cerebral ischemia);脑梗死(cerebral infarction,CI);缺血性脑血管病(ischemic cerebral vascuIar disease)”,以文字和图表形式进行结果的统计和计量分析,描述其分布特征。 结果与结论：内皮祖细胞是成年个体骨髓中的前体细胞,能迁移至外周血并分化为成熟血管内皮细胞,具有良好的增殖潜能,并参与血管内皮修复、缺血器官的新血管形成。根据内皮祖细胞的生物特性与作用,目前国内外研究发现内皮祖细胞能防治脑动脉粥样硬化,参与脑缺血后血管再生,预测评估脑缺血病情,以及对脑出血后具有一定的治疗作用。因此,内皮祖细胞在缺血性脑血管疾病治疗中展现出广泛的应用前景。     

Non-invasive monitoring of transplanted endothelial progenitor cells in diabetic ischemic stroke models

Endogenous endothelial progenitor cells (EPCs) are functionally impaired in hyperglycemia through the p38 MAPK signaling pathway. However, the number and function of transplanted exogenous EPCs in diabetic animals remains unclear. The objectives of this study were to establish a non-invasive imaging strategy to monitor the homing of transplanted EPCs in diabetic stroke mice and to assess the effect of RWJ 67657, an inhibitor of p38 MAPK, on the homing ability of exogenous EPCs. Bone marrow-derived EPCs were labeled in vitro with a multi-functional nanoprobe modified with paramagnetic chelators and fluorophores before being infused into stroke mice. The signal of the nanoprobe reached its peak on day 5 in both magnetic resonance imaging and near-infrared fluorescence imaging after EPC transplantation in wild-type stroke models. The signal enhancement of diabetic stroke models was significantly lower than that of wild-type controls. However, the signal intensity of diabetic stroke models significantly increased after oral administration of RWJ 67657, indicating that more transplanted EPCs migrated to the ischemic brain. Furthermore, the increased exogenous EPCs induced remarkably greater angiogenesis after stroke. These results suggest that this dual-modal imaging strategy is feasible for non-invasively monitoring transplanted cells in vivo.     

YAP is oppositely regulated in i PSC-induced cardiovascular progenitor cell and vascular smooth muscle cell differentiation

AIM: To explore whether YAP protein is important in induced pluripotent stem cell( i PSC)-induced cardiovascular progenitor cell and / or vascular smooth muscle differentiation. METHODS: Using episomal vector based reprogramming,we generated human i PSCs from donor fibroblasts. We used both this i PSCs and human H1 embryonic stem cells to differentiate into vascular smooth muscle cells(VSMCs) through cardiovascular progenitor cells(CVPC). Western blotting,q PCR and immunofluorescence microscopy were used to check the expression of YAP and related genes during this differentiation process. RESULTS: The results showed that i PSCs expressed pluripotent stem cell markers,such as Oct4,Nanog,Sox2,TRA-1-60 and SSEA3,and could form teratoma in SCID mice. YAP was highly expressed in pluripotent stem cells,but dramatically decreased when CVPC differentiation started. YAP gradually increased during CVPC three-day differentiation. The TAZ and YAP binding partner TEAD1,but not TEAD2 and TEAD4,have similar expression pattern in CVPC differentiation. Immunofluorescence result confirmed that YAP was activated and accumulated in nucleus. Interestingly,both YAP and phosphorylated YAP expression decreased to very low level after CVPC differentiated into VSMCs in 7 days. TEAD4 and TAZ also decreased,while TEAD1,TEAD2 and TEAD3 expression did not change during VSMC differentiation. CONCLUSION: YAP and TEAD1 expression increased during CVPC differentiation,while YAP and TEAD4 expression decreased from CVPC to VSMCs differentiation,which suggested YAP might have different function during diverse cell differentiation.     

Oxidative stress impairs endothelial progenitor cell function

Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were identified in 1997. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, recent investigation has focused on the responses of EPCs to oxidant stress and the molecular mechanisms that control redox regulation in these specialized cells. In this review, we discuss the various cell and flow-cytometric techniques used to define and isolate EPCs from circulating blood and the current human and mouse genetic data, which offer insights into redox control in EPC biology and angiogenesis. Finally, we review how EPC responses to oxidant stress may be a critical determinant in maintaining the integrity and function of the cardiovascular system and how perturbations of redox control in EPCs may lead to various human diseases.     

Calpain 3 is a modulator of the dysferlin protein complex in skeletal muscle

Muscular dystrophies comprise a genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and weakness. Two forms of limb-girdle muscular dystrophy, 2A and 2B, are caused by mutations in calpain 3 (CAPN3) and dysferlin (DYSF), respectively. While CAPN3 may be involved in sarcomere remodeling, DYSF is proposed to play a role in membrane repair. The coexistence of CAPN3 and AHNAK, a protein involved in subsarcolemmal cytoarchitecture and membrane repair, in the dysferlin protein complex and the presence of proteolytic cleavage fragments of AHNAK in skeletal muscle led us to investigate whether AHNAK can act as substrate for CAPN3. We here demonstrate that AHNAK is cleaved by CAPN3 and show that AHNAK is lost in cells expressing active CAPN3. Conversely, AHNAK accumulates when calpain 3 is defective in skeletal muscle of calpainopathy patients. Moreover, we demonstrate that AHNAK fragments cleaved by CAPN3 have lost their affinity for dysferlin. Thus, our findings suggest interconnectivity between both diseases by revealing a novel physiological role for CAPN3 in regulating the dysferlin protein complex.     

MicroRNA regulation of endothelial progenitor cell angiogenesis in diabetes

<正>Endothelial progenitor cells(EPCs)are a circulating,bone marrow-derived cell population that participate in both vasculogenesis and vascular homeostasis.Recent studies have shown that EPCs are reduced by~50% in diabetes that correlates inversely with its mortality rate.In addition,EPC angiogenic functions are severely impaired in diabetes.However,the molecular and cellular mechanisms underlying EPC dysfunction are poorly understood.Our current studies have focused on in vitro and in vivo     

外周血内皮祖细胞的诱导培养

目的 探索外周血内皮祖细胞的诱导培养方法。方法 用密度梯度离心分离健康志愿者外周血单个核细胞,EGM-2培养基重悬并在纤连蛋白包被的培养板中进行诱导培养,动态观察贴壁细胞生长状况,免疫荧光技术鉴定内皮祖细胞特性,流式细胞术检测细胞周期分布及其相关表面标志CD34、CD133、CD31、VEGFR2和CD14。结果 贴壁细胞呈细长条状分布,整体形态和生长密度均以第9天最佳,约占外周血单个核细胞的4.62%~5.47%;细胞停留在G0/G1期,增殖指数仅为（1.20±0.18）％;细胞自第5天起即可同时吞噬乙酰化低密度脂蛋白和结合荆豆类凝集素,Ⅷ因子相关抗原于第9天始呈阳性;表面标志CD34、CD133、CD31、VEGFR2和CD14分别为0.19%±0.06%、1.67%±0.52%、61.56%±5.57%、70.29%±7.37%和89.31%±4.11%,共同指示诱导所得细胞属于正在分化的内皮祖细胞。结论 在特定条件下可直接从外周血单个核细胞中诱导培养出内皮祖细胞。     

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。     

The role of endothelial cell lateral junctions during leukocyte trafficking

Summary: An essential function of the inflammatory response is selective targeting of appropriate leukocyte types to a site of infection or injury. The past decade has witnessed an explosion in the level of detail concerning the identification and deciphering of the molecular mechanisms that capture leukocytes from flowing blood and promote leukocyte arrest on the vessel wall. In contrast, less information is known about the migration of adherent blood leukocytes through endothelial cell-to-cell borders (transendothelial migration, TEM) and into the underlying tissues. This article reviews the endothelial-dependent mechanisms that coordinate TEM in peripheral vasculature and highlights the role of certain lateral junctional proteins and protein complexes.     

脉冲电刺激对血管内皮细胞与其祖细胞黏附的影响

为探索脉冲电刺激下血管内皮细胞与内皮祖细胞(EPC)之间黏附强度的改变,诱导培养外周血EPC,荧光标记后与单层血管内皮细胞共培养,固定电压和频率为5 V和5 Hz,选择1、3、6、9 ms的脉宽对其进行干预,持续刺激24 h后检测贴壁EPC的荧光强度,以荧光比率衡量。结果显示,与对照组相比,3 ms刺激组荧光比率即显著增高,随着脉宽延长,6 ms组达到最大值,但9 ms刺激组却显著下降,提示适宜脉冲电刺激有利于血管内皮细胞与EPC之间的黏附,为电刺激促进血管新生的研究提供新的理论依据。