A prototype tissue engineered blood vessel using amniotic membrane as scaffold

In this study, we used amniotic membrane (AM), a natural extracellular matrix, as a scaffold for the fabrication of tissue engineered blood vessels (TEBVs). The inner surface of the denuded glutaraldehyde cross-linked AM tube was endothelialized with porcine vascular endothelial cells (ECs) and subjected to a physiological (12 dynecm(-2)) shear stress (SS) for 2 and 4 days. The results showed that after applying SS, an intact EC monolayer was maintained in the lumen surface of the TEBV. The ECs were aligned with their long axis parallel to the blood flow. The immunofluorescent microscopy showed that the intercellular junctional proteins, PECAM-1 and VE-cadherin, were surrounding the EC periphery and were better developed and more abundant in SS-treated TEBVs than the static controls. The Western blot indicated that the expressions of PECAM-1 and VE-cadherin were increased by 72 ± 9% and 67 ± 7%, respectively, after shear stress treatment. The distribution pattern of integrin β1 was mainly at the interface of ECs and AM in static TEBVs but it was extended to the cell-cell junctions after SS treatment. The SS promoted the expression of integrin α(v)β(3) without altering its distribution in TEBV. The results suggest that glutaraldehyde cross-linked AM tube can potentially be used as a scaffold biomaterial for TEBV fabrication. Most importantly, the use of an AM tube shortened the TEBV fabrication.     

Dynamics of T cells on endothelial layers aligned by nanostructured surfaces

In this work, well-aligned endothelial cell (EC) layers were prepared by culturing ECs on surfaces containing nanoscale ridges/grooves fabricated by UV-assisted capillary force lithography. Then, the dynamics of T cells on well-aligned ECs were compared with that on randomly oriented ECs cultured on flat surfaces. With this experimental setting, we demonstrated for the first time that EC alignment is important for the regulation of transendothelial migration (TEM) of T cells, a critical step for leukocyte infiltration; T cells preferentially underwent TEM at the junctions surrounded by more than three ECs only if ECs surrounding those junctions were poorly aligned. As a result, TEM of T cells occurred more quickly and frequently on randomly oriented ECs cultured on flat surfaces than on well-aligned ECs cultured on nanostructured surfaces. This result will suggest a new strategy for the design of synthetic small diameter vascular grafts and extend our current knowledge of leukocyte dynamics on an inflamed endothelium.     

EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury

The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316-324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up-regulation may be a critical component in the reorganization of the vascular bed in response to angiogenic stimuli.     

VE-statin/Egfl7 siRNA inhibits angiogenesis in malignant glioma in vitro

This study investigated the role of VE-statin/Egfl7 and its mechanism in angiogenesis in malignant glioma. Transwell culture plates were used to establish an U251-HUVEC co-culture system, which was used to mimic the interaction between malignant glioma and endothelial cells. Lentiviral vectors expressing VE-statin/Egfl7 siRNA were constructed, and U251 cells and HUVECs were transfected to inhibit VE-statin/Egfl7 expression. The proliferation, adherence, migration, and lumen formation of endothelial cells were assayed to investigate the influence of VE-statin/Egfl7 on angiogenesis in malignant glioma in vitro. Data showed that HUVEC growth was temporarily slowed after silencing the VE-statin/Egfl7 gene but rapidly returned to normal. Although endothelial cell migration was not influenced, cell adherence was markedly inhibited. Furthermore, the endothelial cells failed to generate a capillary-like lumen after VE-statin/Egfl7 gene silencing. Therefore, it can be concluded that VE-statin/Egfl7 may regulate the adherence of endothelial cells, thus playing an important role in endothelium-induced lumen formation during angiogenesis in malignant glioma.     

Establishment of the vascular intimal model in vitro and its application in studying atherosclerosis]

An endothelial culture model in vitro mimicking the vascular intima in vivo was designed which was composed of an upper and a lower well separated by a layer of amnion membrane, and upon which endothelial cells (ECs) isolated from the human umbilical vein were cultured. The upper well, the subendothelial amnion, and the lower well were analogical to the vascular lumen, the subendothelial tissue and the extravascular space, respectively. In comparison with the ECs cultured on the plastic, dishes, ECs cultured on the amnion membrane maintained more morphologic features as in vivo and could be cultured for up to 15 days without apparent detachment. Monocytes, loaded in the upper wells, were able to adhere to the cytoplasmic membrane of ECs. Furthermore, with the presence of the chemotactic factor (fMLP) in the lower well, monocytes showed active migrating ability passing through the EC junctions. If LDL (100 micrograms/ml) was added in the media simultaneously, monocytes might be aggregated beneath the subendothelial space and some of them took an foamy appearance. In conclusion, the culture model is of value in the study of experimental inflammation and atherosclerosis.     

“Deep-media culture condition” promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro

In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a “deep-media culture conditions.” Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.     

A Dynamically Cultured Collagen/Cells-Incorporated Elastic Scaffold for Small-Diameter Vascular Grafts

Abstract

There is an essential demand for tissue-engineered autologous small-diameter vascular grafts, which offer temporary supports and guides for vascular tissue organization, repair and remodeling. This study reports on the effect of collagen/smooth muscle cells (SMCs) mixtures under dynamic cultures and SMC-endothelial cell (ECs) co-culture on cell proliferation, uniform cell distribution, extracellular matrix deposition, and endothelial cells monolayer formation in tissue-engineered tubular arterial constructs of 4 mm inner diameter. Rabbit aortic SMCs were infiltrated with collagen solution in poly(L-lactide-co-?-caprolactone) (PLCL) scaffolds under vacuum to form collagenous gel and subjected to dynamic strain by culturing them in a dynamic perfusion bioreactor. The construct lumen was subsequently seeded with ECs and experiments were completed to create ECs–SMCs co-culture constructs. The collagen/SMCs incorporated elastic scaffold cultured under dynamic culture conditions promoted matrix deposition, leading to the development of tissue-engineered vascular constructs, and induced SMC to have more uniform cell distribution. Scanning electron microscopic examination and von Willebrand Factor staining demonstrated the presence of ECs spread over the lumen. Quantitative analysis of elastin contents demonstrated that the engineered vessels acquired similar elastin contents as native arteries. The collagen/SMCs/ECs incorporated PLCL scaffolds under dynamic culture conditions can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.     

VE-statin/Egfl7 expression in malignant glioma and its relevant molecular network

This study investigated VE-statin/Egfl7 expression and its role and regulatory mechanism in malignant glioma progression. Forty-five paraffin-embedded glioma (grade I-II: n=24; grade III-IV: n=21) were examined. VE-statin/Egfl7 protein expression was detected via immunohistochemistry, and its correlation with pathological grade was evaluated. Three-dimensional cell culture was then performed to investigate the influence of VE-statin/Egfl7 on the angiogenesis of umbilical vein endothelial cells. Microarray detection was used to molecularly profile VE-statin/Egfl7 and relevant signaling pathways in malignant glioma (U251 cells). Data showed that VE-statin/Egfl7 protein was mainly expressed in the cytoplasm of cancer and vascular endothelial cells and was significantly related to the degree of malignancy (t=4.399, P<0.01). Additionally, VE-statin/Egfl7 expression was low in certain gray-matter neurons but undetectable in glial cells. VE-statin/Egfl7 gene silencing significantly inhibited angiogenesis in umbilical vein endothelial cells. The following microarray results were observed in VE-statin/Egfl7-silenced U251 cells: 1) EGFR family members showed the highest differential expression, accounting for 5.54% of differentially expressed genes; 2) cell survival-related signaling pathways changed significantly; and 3) the integrin ανβ3 signaling pathway was markedly altered. Thus, malignant glioma cells and glioma vascular endothelial cells highly express VE-statin/Egfl7, which is significantly correlated with the degree of malignancy. Moreover, VE-statin/Egfl7 plays an important role in glioma angiogenesis. Microarray results indicate that VE-statin/Egfl7 may regulate EGFR and integrins to influence the FAK activity of downstream factors, triggering the PI3K/Akt and Ras/MAPK cascades and subsequent malignant glioma development.     

Molecular Pathways Governing Development of Vascular Endothelial Cells from ES/iPS Cells

Assembly of complex vascular networks occurs in numerous biological systems through morphogenetic processes such as vasculogenesis, angiogenesis and vascular remodeling. Pluripotent stem cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells can differentiate into any cell type, including endothelial cells (ECs), and have been extensively used as in vitro models to analyze molecular mechanisms underlying EC generation and differentiation. The emergence of these promising new approaches suggests that ECs could be used in clinical therapy. Much evidence suggests that ES/iPS cell differentiation into ECs in vitro mimics the in vivo vascular morphogenic process. Through sequential steps of maturation, ECs derived from ES/iPS cells can be further differentiated into arterial, venous, capillary and lymphatic ECs, as well as smooth muscle cells. Here, we review EC development from ES/iPS cells with special attention to molecular pathways functioning in EC specification.     

Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.

We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino-terminal signal peptide and two centrally located EGF-like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF-like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7.     

Local thermal injury induces general endothelial cell contraction through p38 MAP kinase activation

Endothelial cells (ECs) of thin‐walled blood vessels form a barrier between blood and tissue. As a response to inflammation, the EC junctions widen and gaps form, resulting in compromised barrier functions. Although the mechanisms behind the establishment of these changes are still incompletely understood, one known reason is actomyosin‐dependent actin rearrangement. Here, by using atomic force microscopy and a combination of confocal microscopy methods, we are the first to report that thermal injury induces general venular hyperpermeability and that serum from burned rats induces EC actin rearrangement, contraction, as well as tight‐junction damage. Inhibition of the p38 mitogen‐activated protein kinase (p38MAPK) largely ameliorates resulting vascular dysfunction by significantly reducing EC stress‐fiber formation, contraction, volume changes and tight‐junction damage, thereby greatly reducing the appearance of EC gaps. The findings may be of importance for the design of future pharmacotherapies aiming to ease the severe general vascular dysfunction that follows extensive burns.     

The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.     

兔不同血管段内皮细胞的扫描电镜观察

目的：观察兔不同血管段内皮细胞(EC)的形态特征，为兔血管的临床和实验研究提供资料。方法：动物灌注固定，取出相应血管，扫描电镜观察。结果：EC一般呈梭形，核区较隆起，长轴与血流的方向一致。在腹主动脉相邻细胞之间有纵行的沟，细胞连接处有较矮的嵴。在肺动脉有较丰富粗短的微绒毛和长短不一的桥样结构。在主动脉瓣和二尖瓣，细胞呈较大的圆形或椭圆形，微绒毛较矮，密度不均。在主动脉弓细胞形态多样。在下腔静脉，细胞呈窄长的梭形。结论：EC的形态、排列、微绒毛的长度和密度，桥样结构的多少等均随部位的不同而有差异，切应力可能是造成这些差异的重要因素。     

First steps of tumor-related angiogenesis.

We present morphological data of the early steps of tumor-induced angiogenesis and show the distribution of the three main components of the basal lamina (BL), laminin, collagen IV, and fibronectin during these early processes. Tumor cells of a line of BSp73 AS a nonmetastasizing tumor isolated from a pancreatic adenocarcinoma were injected subcutaneously into the back of BDX rats. Two days after tumor inoculation, the BL of the dilated mother vessels around the whole circumference of the vessel has either disappeared, become fragmented, or developed several successive layers. By immunoelectron microscopy, we demonstrate that the fragmented and multilayered BL is strongly stained for laminin and collagen IV but less strongly for fibronectin. Around the surface of the dilated mother vessels which are free of any detectable BL material (by electron microscopy standards), we can see accumulation of all three components in the connective tissue. Simultaneously with the alteration of the BL, the proliferation of the endothelial cells (EC) and the pericytes and the migration of the EC from the wall of the mother vessel have started. EC migration begins in two different ways. Either one EC migrates from the wall of the mother vessel into the surrounding connective tissue, or two or more EC form nearly parallel processes toward the connective tissue. The tips of these processes are connected by intracellular junctions. Around the cellular protrusions of these cells material of the BL deposited into the nearby connective tissue can be observed neither by conventional nor by immunoelectron microscopy. During the outgrowth and migration, the EC remain in contact via junctions with the EC of the original vessel. When migration during which the EC retain their polarization continues, a slit-like lumen forms immediately between the migrating EC. This lumen always remains in direct connection with the lumen of the mother vessel. It is sealed at its border by intercellular junctions. Such junctional complexes can develop a length (in sections) of several hundred micrometers. A BL detectable in the electron microscope can neither be found around the tip of the migrating EC nor around young capillaries not yet surrounded by pericytes. By immunoelectron microscopy, however, only the cellular protrusions at the tip of migrating EC are free of deposited material of the BL. The basal surface of longer (new) capillaries is covered by a continuous layer of amorphous material.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Cardiac Fibroblasts Support Endothelial Cell Proliferation and Sprout Formation but not the Development of Multicellular Sprouts in a Fibrin Gel Co-Culture Model

A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mechanical functions, but to date have not been sufficiently studied for their role in angiogenesis. To study CFs role in angiogenesis, we co-cultured different concentrations of various cell types in fibrin hemispheres with appropriate combinations of their specific media, to determine the optimal conditions for EC growth and sprout formation through DNA analysis, flow cytometry and immunohistology. ECs proliferated best when co-cultured with CFs and analysis of immunohistological images demonstrated that ECs formed the longest and most numerous sprouts with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs. Moreover, these effects were dependent on the ratio of support cell to EC in co-culture. Overall, CFs provide a good support system for EC proliferation and sprout formation; however, MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.     

Cellular engineering of vascular bypass grafts: Role of chemical coatings for enhancing endothelial cell attachment

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5–6 mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patient's own cells. Currently, to be effective, high-density seeding regimens have to be undertaken. This is because endothelial cells (ECs) are washed off the graft lumen once exposed to physiological blood flow. EC attachment has been shown to be significantly improved by pre-coating with substances known to attach ECs selectively. The review examines the various types of coating and bonding technology used to date to enhance endothelial cell attachment onto the surface of prosthetic vascular bypass grafts.     

骨髓间充质干细胞在肿瘤血管生成过程中的作用

目的 研究人骨髓间充质干细胞(hMSC)在肿瘤血管生成过程中的作用.方法 通过梯度密度离心法及贴壁筛选法分离、培养hMSC;利用pLEGFP-N1逆转录病毒载体获得增强型绿色荧光蛋白(EGFP)标记的hMSC(hMSC-EGFP);流式细胞仪检测hMSC-EGFP的免疫表型及分化能力;通过建立BALB/C裸鼠实体瘤模型(分别皮下接种乳腺癌细胞系MCF-7及hMSC-EGFP细胞与MCF-7混悬液)来观察hMSC在肿瘤血管生成过程中的作用;检测肿瘤细胞和内皮细胞条件培养基对hMSC细胞生长和迁移的影响;体外诱导hMSC向内皮细胞分化,观察其对人脐静脉内皮细胞(HUVEC)迁移的影响.结果 hMSC-EGFP与hMSC形态相似,均呈成纤维细胞样;二者具有相似的免疫表型,在条件基质作用下,均能被诱导分化为成骨细胞及脂肪细胞;hMSC能促进肿瘤血管生成,单纯接种MCF-7组肿瘤平均血管密度为5.33±1.42,混悬液组为13.67±1.53,差异有统计学意义(P<0.05);大部分血管是由hMSC植入体内引起的宿主源性血管新生,只有少数血管的内皮细胞是由hMSC植入体内分化而来的;肿瘤细胞和内皮细胞能够通过其旁分泌作用促进hMSC的生长和迁移;经内皮诱导2周后,hMSC呈CD31阳性;hMSC能促进HUVEC的迁移.结论 MSC具有促进肿瘤血管生成的作用.     

阿托伐他汀对高胆固醇血症大鼠侧支血管生长的影响

目的　探讨阿托伐他汀对高胆固醇血症大鼠侧支血管生长的影响。 方法　28只成年SD大鼠,给予高脂饮食8周,建立结扎股动脉诱导的高胆固醇血症大鼠侧支血管生长模型。随机将动物分为单纯股动脉结扎组（L组）、高胆固醇血症+股动脉结扎组（HL组）和阿托伐他汀（0.3 mg·kg-1·14 d-1,腹腔注射）+高胆固醇血症+股动脉结扎组（AL组）。存活7 d后,采用血管造影,HE染色和共聚焦免疫荧光术,观察高胆固醇血症情况下,阿托伐他汀应用对侧支血管生长的作用以及重要的促侧支血管生长分子在侧支血管表达模式的变化。 结果　与L组比较,HL组的侧支血管数目减少,侧支血管生长受到损害,表现为血管内膜过度增生,中膜明显增厚,导致血管腔狭窄,且血管壁细胞增殖和外膜炎症细胞减少;AL组应用了阿托伐他汀,侧支血管的生长较HL组明显改善,发育为管腔较大的侧支血管,血管壁细胞的增殖和外膜炎症细胞增多,侧支血管数目增加。其侧支血管数目,血管横截面积和免疫荧光强度差异具有统计学意义（P<0.05）。 结论　高胆固醇血症损害大鼠后肢侧支血管的生长;阿托伐他汀可促进血管壁细胞的增殖和外膜巨噬细胞的增多,从而改善和恢复侧支血管的生长。     

内皮细胞的异质性

内皮细胞是一衬覆于血管腔内表面的异质性群体。不同种属、不同管径、不同组织的血管内皮细胞之间及肿瘤性血管内皮细胞之间，在结构、功能、表面分子等方面表型各异。内皮细胞的异质性随时空而变化，其机制可能与细胞基因修饰的内在因素和/或细胞外微环境诱导的外在因素相关。