血流剪切应力与内皮细胞命运

血管发育和稳态维持以及血管病理进程均依赖于血管内衬的内皮细胞(endothelial cells,ECs)。ECs与血液直接接触并受到血流剪切应力的作用,血流剪切应力通过调控ECs的发育和细胞命运等生物学过程来调节血管稳态。ECs命运转化主要包括生理条件下通过内皮-造血干细胞转化(endothelial-to-hematopoietic transition,EHT)产生造血干/祖细胞进入循环系统,以及病理条件下经内皮-间充质转化(endothelial-to-mesenchymal transition,EndMT)形成间充质细胞参与血管狭窄等,这两种命运转化的共同特点之一是细胞间黏附分子和黏附力的破坏。本文旨在概述血流剪切应力调控内皮细胞EHT和EndMT的分子机制,将提升对心血管发育和相关疾病发生机制的理解。

造血干细胞发生的研究进展

哺乳动物个体的发育起源于受精卵的形成，受精卵是个体的第1个全能干细胞。受精卵的分裂和分化逐步形成相对定向的胚胎阶段的多种干细胞及组织特异性的成体干细胞,包括成体造血干细胞（hematopoietic stem cells，HSCs）在内。已有充分的证据表明在人和小鼠中，HSC是成体内各系造血细胞的来源，单个HSC即可在合适的宿主体内重建整个造血系统。

Prokineticin 2/Bv8 is expressed in Kupffer cells in liver and is down regulated in human hepatocellular carcinoma

AIM： TO study the implication of prokineticin 1 （PKI/EGVEGF） and prokineticin 2 （PK2/13v8） in hepatocellular carcinoma angiogenesis.METHODS： The gene induction of PK1/EG-VEGF and PK2/Bv8 was investigated in 10 normal, 28 fibrotic and 28 tumoral livers by using real time PCR. Their expression was compared to the expression of VEGF （an angiogenesis marker）, vWF （an endothelial cell marker） and to CD68 （a monocyte/macrophage marker）. Furthermore, the rnRNA levels of PK1/EG-VEGF, PK2/Bv8, prokineticin receptor 1 and 2 were evaluated by real time PCR in isolated liver cell populations. Finally, PK2/Bv8 protein was detected in normal liver paraffin sections and in isolated liver cells by immunohistochernistry and immunocytochemistry.RESULTS： PK2/Bv8 mRNA but not PK1/EG-VEGF was expressed in all types of normal liver samples examined. In the context of liver tumor development, we reported that PK2/13v8 correlates only with CD68 and showed a significant decrease in expression as the pathology evolves towards cancer. Whereas, VEGF and vWF mRNA were significantly upregulated in both fibrosis and HCC,as expected. In addition, out of all isolated liver cells examined, only Kupffer cells （liver resident macrophages） express significant levels of PK2/Bv8 and its receptors, prokineticin receptor 1 and 2.CONCLUSION： In normal liver PK2/Bv8 and its receptors were specifically expressed by Kupffer cells. PK2/Bv8 expression decreased as the liver evolves towards cancer and did not correlate with HCC angiogenesis.

Proliferation of endothelial cell on polytetrafluoroethylene vascular graft materials carried VEGF gene plasmid

Objective： To investigate whether vascular endothelial growth factor （VEGF） gene plasmid carried by polytetrafluoroethylene （PTFE） vascular graft materials could transfect endothelial cells （ECs） and promote their growth. Methods： PTFE vascular graft materials carried with pCDI-hVEGF121, pCDI or pEGFP were incubated in Tris-buffer solution and the values of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs derived from human umbilical vein were seeded on the pCDI-hVEGF121/pCDI/pEGFP-PTFE materials or tissue culture plates, ECs numbers were counted and VEGF protein concentrations at different time were measured by enzyme-linked immunoadsorbent assay method. Green fluorescent protein （GFP） expression in ECs on pEGFP-PTFE materials was examined with fluorescence mi- croscopy. Results： The controlled release curve showed that the gene released from PTFE materials was rapid within 8 h, then slowed down and that the gene released continuously even after 72 h. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGFI21-PTFE materials were higher than those ofpCDI or pEGFP-PTFE materials （P〈0.05）. VEGF protein concentration of pCDI-hVEGF121-PTFE materials was higher than that of pC DI or pEGFP-PTFE materials at 6, 24, 72 and 120 h （P〈0.01）. GFP expression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy. Conclusion： PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth.

IMPAIRED ANGIOGENESIS FOLLOWING HIND-LIMB ISCHEMIA IN DIABETES MELLITUS MICE

Objective To investigate vascular endothelial growth factor(VEGF) and its signaling pathway spontaneous response in type 2 diabetes mellitus(T2DM) mice to surgery-induced hind-limb ischemia.Methods Sixty mice were randomly divided into two groups,one was fed with normal chow as control,and another was fed with high fat diet to induce T2DM.Fourteen weeks later,mice were surgically induced to hind-limb ischemia.Blood flow restoration was monitored with laser Doppler perfusion imaging.Tibialis anterior muscle was collected after 3 days of hind-limb ischemia.VEGF mRNA and protein expressions were analyzed using real-time PCR and ELISA;expressions of VEGF downstream signal molecules and receptors were analyzed using Western blotting and RT-PCR,respectively.Results Perfusion recovery 10,20,30 days after ischemia was significantly attenuated in T2DM compared with control group(P<0.05).T2DM impaired VEGF signaling pathway although VEGF levels increased in T2DM group.After ischemia,T2DM group had a comparable increase in VEGF expression compared with control group,but still had an impaired VEGF signaling pathway.Conclusion VEGF signaling pathway is abnormal in T2DM mice,although VEGF had a response to the ischemic stimulation.

低氧通过上调Wnt5a促进人胚胎干细胞向生血内皮细胞分化

胚胎的早期发育是在低氧条件下进行的,低氧环境在胚胎血管发生及造血发育中起着重要作用,低氧条件能促进胚胎干细胞在体外向内皮细胞和造血细胞的分化,但低氧条件对造血细胞产生的具体作用及相应机制尚不清楚.本研究利用人ES细胞向造血祖细胞定向分化体系,发现低氧环境可以促进CD31+TIE2+造血内皮祖细胞的产生,2天后造血内皮祖细胞开始表达终生造血基因.进一步研究发现,低氧能够上调Wnt5a的表达,干涉Wnt5a能够抑制低氧环境对生血内皮细胞分化的促进作用.在正常氧环境下加入Wnt5a产生促进生血内皮细胞分化的效应,该效应与低氧处理促进生血内皮细胞产生的作用相似.本研究首次证明了低氧通过上调Wnt5a的表达促进人ES细胞向生血内皮细胞的分化,为ES细胞向生血内皮细胞的分化及造血祖细胞分化的研究提供了新的线索.

造血干细胞发育的分子机制

造血干细胞是一群具有自我更新及分化能力的多能干细胞,可以分化为所有类型的成熟血细胞.造血干细胞的发育过程受到多种转录因子及信号通路的精密调控,任何调控失衡都将导致严重的发育缺陷或者重大疾病.本文系统总结了脊椎动物(小鼠及斑马鱼)造血干细胞发育过程中发挥关键作用的信号通路及转录因子,不仅有助于理解造血干细胞发生的分子机制,而且对完善调控网络以及促进基础向临床转化具有一定的理论指导意义.

Recombinant human Flt3 ligand exerts both direct and indirect effects on hematopoiesis

OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells. METHODS: Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL. CONCLUSIONS: rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.

Role of precoating in artificial vessel endothelialization

As the progress of vascular surgery, artificial vessels have become the substitute for large and middle diameter vessels but have not for small diameter ones owing to thrombogenesis and occlusion within a short period of time after being applied. Artificial vessel endothelialization is one of the ideal methods to resolve such issue and has been improved continuously since Herring in 1978 put forward this term in the first time and utilized vascular endothelial cells (ECs) harvested from living animals to perform the test of artificial vessel endothelialization. However, human endothelial cells show little adhesion to the currently available vascular graft materials and some expanded polytetrafluoroethylene (ePTFE) grafts have shown only 10%+/-7% endothelial cell attachment rate (ECA, ie, attachment of ECs when incubated in vitro). Moreover, when the graft is exposed to pulsatile blood flow, a high proportion of cells are washed off from the lumen. Maximum cell loss occurs in the first 30-45 min after exposure to pulsatile flow, with up to 70% of cells lost. After that, a slower exponential loss occurs over the next 24 h. The lack of retention of cells could be partly overcome by sodding, but other techniques, involving engineering the lumen to improve ECA and endothelial cell retention rate (ECR, ie, retention of ECs when the grafts are exposed to pulsatile flow) have been developed. These include shear stress preconditioning, electrostatic charging and, above all, most successfully to date, precoating with EC specific adhesive glues that are mostly found in the extracellular basement membrane of blood vessels. The commonest are chemical coatings, preclotting, chemical bonding, and surface modifications.

On signaling pathways: hematopoietic stem cell specification from hemogenic endothelium

Hematopoietic stem cells(HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells(PSCs-embryonic stem cells(ESCs) and induced pluripotent stem cells(i PSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.