Coordinating cell behaviour during blood vessel formation

The correct development of blood vessels is crucial for all aspects of tissue growth and physiology in vertebrates. The formation of an elaborate hierarchically branched network of endothelial tubes, through either angiogenesis or vasculogenesis, relies on a series of coordinated morphogenic events, but how individual endothelial cells adopt specific phenotypes and how they coordinate their behaviour during vascular patterning is unclear. Recent progress in our understanding of blood vessel formation has been driven by advanced imaging techniques and detailed analyses that have used a combination of powerful in vitro, in vivo and in silico model systems. Here, we summarise these models and discuss their advantages and disadvantages. We then review the different stages of blood vessel development, highlighting the cellular mechanisms and molecular players involved at each step and focusing on cell specification and coordination within the network.     

外源性端粒酶基因对人脐静脉内皮细胞的影响

为了观察外源性端粒酶逆转录酶基因(hTERT)在人脐静脉血管内皮细胞(HUVEC)的表达及对细胞功能和生长的影响。采用逆转录病毒载体转导的方法,将hTERT基因转入HUVEC,检测基因转导后内皮细胞端粒酶的活性和生物学特性。结果发现hTERT转导后细胞端粒酶表达阳性,未转导的亲代细胞为阴性;转导细胞的体外生存时间延长但未永生化,而内皮细胞黏附分子表达的功能未受影响。     

Mechanical Cell-Matrix Feedback Explains Pairwise and Collective Endothelial Cell Behavior In Vitro

In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a) the contractile forces that endothelial cells exert on the ECM, (b) the resulting strains in the extracellular matrix, and (c) the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.     

Incorporation of adult organ-derived endothelial cells into tumor blood vessel

In this study, we attempted to assess the incorporable potential of vascular endothelial cells derived from adult organ blood vessels into tumor blood vessels. Two kinds of adult organ-derived vascular endothelial cells, human aorta endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC), were administered into murine tumors inoculated to SCID mice. Many human blood vessel networks were visualized in the murine tumors. These cells in solid tumor not only survived and proliferated, but also incorporated into tumor endothelium. These results suggest that adult organ-derived vascular endothelial cells possess the potential to form the neovascular network in various tissues such as vascular endothelial progenitor-like cells in vivo. We propose that these cells can be regarded as a congenic (autologous) vector for vascular regeneration cell therapy and tumor vascular targeting gene therapy.     

Contact-inhibited chemotaxis in de novo and sprouting blood-vessel growth

Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully formed blood vessels) organize into a vessel network (vasculogenesis), or by sprouting or splitting of existing blood vessels (angiogenesis). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier-Graner-Hogeweg model (also called Cellular Potts Model) simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller-Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin-mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface-normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell-cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.     

血管内皮细胞和心脏组织块的立体培养

本文旨在对比研究二维平面与三维立体培养模式下,内皮细胞和心脏组织形态学的差异。采用胶内、胶上、三明治模式、玻片培养小室模型等多种I型胶原立体培养模型,通过免疫荧光技术及显微形态学观察组织和细胞的生长情况。在二维平面培养中,原代心脏血管内皮细胞呈铺路石样排列;而在三维胶原培养模式中,内皮细胞呈长梭状形态,并迁入胶原培养介质中,和体内血管新生及血管生成过程中的内皮细胞活化表型相似。加入血管内皮生长因子(vascular endo- thelial growth factor VEGF)能增强内皮细胞管状结构的形成。在三维胶原中,心脏组织块生长良好,迁出的细胞将相邻组织块连接起来,组织块有自发的搏动。本工作表明,改进的薄层胶原培养、玻片培养小室模型和动脉条模型是较好的研究血管生成和血管新生的工具。在三维培养的情况下,内皮细胞通过空间增殖、迁移和锚定,可形成管状结构,比二维平面培养更适合用于血管新生的研究。不同的立体培养模型可用于不同目的的研究。     

Induction of vasculogenesis and hematopoiesis in vitro.

Despite a large number of investigations of embryonic vascular development, in particular in avian embryos, the conditions under which the endothelial and hematopoietic cell lineages emerge remain unknown. As we demonstrate here, both endothelial and hematopoietic cells can be induced by treatment of dissociated quail epiblast with fibroblast growth factors in vitro. These cells aggregate in characteristic blood islands. In long-term culture, the induced endothelial cells gave rise to vascular structures in vitro, i.e. vasculogenesis. No induction was observed in the absence of fibroblast growth factors, and other growth factors like TGF-beta, TGF-alpha and EGF were not capable of inducing blood island formation. Thus, the dissociated quail epiblast provides a remarkably simple test system to investigate cell lineage diversification in higher vertebrates.     

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.     

A Titin mutation defines roles for circulation in endothelial morphogenesis

Morphogenesis of the developing vascular network requires coordinated regulation of an extensive array of endothelial cell behaviors. Precisely regulated signaling molecules such as vascular endothelial growth factor (VEGF) direct some of these endothelial behaviors. Newly forming blood vessels also become subjected to novel biomechanical forces upon initiation of cardiac contractions. We report here the identification of a recessive mouse mutation termed shrunken-head (shru) that disrupts function of the Titin gene. Titin was found to be required for the initiation of proper heart contractions as well as for maintaining the correct overall shape and orientation of individual cardiomyocytes. Cardiac dysfunction in shrunken-head mutant embryos provided an opportunity to study the effects of lack of blood circulation on the morphogenesis of endothelial cells. Without blood flow, differentiating endothelial cells display defects in their shapes and patterns of cell-cell contact. These endothelial cells, without exposure to blood circulation, have an abnormal distribution within vasculogenic vessels. Further effects of absent blood flow include abnormal spatial regulation of angiogenesis and elevated VEGF signaling. The shrunken-head mutation has provided an in vivo model to precisely define the roles of circulation on cellular and network aspects of vascular morphogenesis.     

血管内皮细胞发育及分子机制

心血管系统是胚胎发育中最先形成的器官之一, 为机体提供营养成分和氧气。血管发育包括两部分, 一是内皮祖细胞(Angioblast)聚集形成血管原基(Vasculogenesis), 二是从已有血管形成新的血管分支(Angiogenesis)。此后由初级内皮细胞管召集平滑肌细胞形成功能性血管(Vessel maturation)。内皮祖细胞起源途径包括：由Flk1阳性中胚层细胞到成血成血管细胞(Hemangioblast)到血管内皮祖细胞; 或由Flk1阳性中胚层细胞直接到血管内皮祖细胞。Flk1阳性中胚层细胞受到vegf、flk1、cloche、lycat、etsrp等关键基因或信号通路的调节, 其中核心问题是原肠期中胚层如何形成Flk1阳性中胚层细胞及进一步分化成血管内皮祖细胞和成血血管细胞。文章集中评述内皮祖细胞发育、分化及其分子遗传调控机制, 并展望本领域未来发展方向。     

Human primary endothelial cells stimulate human osteoprogenitor cell differentiation.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly endothelial cells that may be pivotal members of a complex interactive communication network in bone. While cell cooperation was previously established between Human OsteoProgenitor cells (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) the aim of our study was to investigate if this interaction is specific to Human Endothelial cell types (ECs) from different sources. Osteoblastic cell differentiation analysis performed using different co-culture models with direct contact revealed that Alkaline Phosphatase (Al-P) activity was only increased by the direct contact of HOP with human primary vascular endothelial cell types including endothelial precursor cells (EPCs) isolated from blood cord, endothelial cells from Human Saphen Vein (HSV) while a transformed cell line, the Human Bone Marrow Endothelial Cell Line (HBMEC) did not modify osteoblastic differentiation of HOP. Because connexin 43, a specific gap junction protein, seemed to be involved in HUVEC/HOP cell cooperation, expression by RT-PCR and immunocytochemistry of this gap junctional protein was investigated in EPCs, HSV and HBMEC. Both endothelial cells are positive to this protein and the disruption of gap junction communication using 18alpha-glycyrrhetinic acid treatment decreased the positive effect of these endothelial co-cultures on HOP differentiation as was previously demonstrated for HUVEC and HOP co-cultures. These data seem to indicate that this cross talk between HOP and ECs, through gap junction communication constitutes an additional concept in cell differentiation control.     

Doxazosin inhibits human vascular endothelial cell adhesion, migration, and invasion

The quinazoline-derived alpha1-adrenoceptor antagonists, doxazosin and terazosin have been recently shown to induce an anoikis effect in human prostate cancer cells and to suppress prostate tumor vascularity in clinical specimens [Keledjian and Kyprianou, 2003]. This study sought to examine the ability of doxazosin to affect the growth of human vascular endothelial cells and to modulate vascular endothelial growth factor (VEGF)-mediated angiogenesis. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro model to determine the effect of doxazosin on cell growth, apoptosis, adhesion, migration, and angiogenic response of endothelial cells. The effect of doxazosin on cell viability and apoptosis induction of human endothelial cells, was evaluated on the basis of trypan blue and Hoechst 33342 staining, respectively. Doxazosin antagonized the VEGF-mediated angiogenic response of HUVEC cells, by abrogating cell adhesion to fibronectin and collagen-coated surfaces and inhibiting cell migration, via a potential downregulation of VEGF expression. Furthermore there was a significant suppression of in vitro angiogenesis by doxazosin on the basis of VEGF-mediated endothelial tube formation (P < 0.01). Fibroblast growth factor-2 (FGF-2) significantly enhanced HUVEC cell tube formation (P < 0.01) and this effect was suppressed by doxazosin. These findings provide new insight into the ability of doxazosin to suppress the growth and angiogenic response of human endothelial cells by interfering with VEGF and FGF-2 action. This evidence may have potential therapeutic significance in using this quinazoline-based compound as an antiangiogenic agent for the treatment of advanced prostate cancer.     

Cellular repressor of E1A-stimulated genes regulates vascular endothelial cell migration by the ILK/AKT/mTOR/VEGF(165) signaling pathway

The migration of vascular endothelial cells plays a critical role in a variety of vascular physiological and pathological processes, such as embryonic development, angiogenesis, wound healing, re-endothelialization, and vascular remodeling. This study clarified the role and mechanism of a new vascular homeostasis regulator, Cellular repressor of E1A-stimulated genes (CREG), in the migration of primary human umbilical vein endothelial cells (HUVECs). A wound healing assay and transwell migration model showed that upregulation of CREG expression induced HUVEC migration and it was positively correlated with the expression of vascular endothelial growth factor. Furthermore, wild type integrin-linked kinase reversed the poor mobility of CREG knock-down HUVECs; in contrast, kinase-dead integrin-linked kinase weakened the migration of HUVECs. We also studied the effect of CREG on HUVEC migration by the addition of an mTOR inhibitor, recombinant vascular endothelial growth factor₁₆₅, neutralizing antibody of vascular endothelial growth factor₁₆₅ and AKT siRNA, and we concluded that CREG induces endothelial cell migration by activating the integrin-linked kinase/AKT/mTOR/VEGF₁₆₅ signaling pathway.     

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF’s blood vessel forming potential prior to clinical implantation.     

Modelling the Role of Angiogenesis and Vasculogenesis in Solid Tumour Growth

Recent experimental evidence suggests that vasculogenesis may play an important role in tumour vascularisation. While angiogenesis involves the proliferation and migration of endothelial cells (ECs) in pre-existing vessels, vasculogenesis involves the mobilisation of bone-marrow-derived endothelial progenitor cells (EPCs) into the bloodstream. Once blood-borne, EPCs home in on the tumour site, where subsequently they may differentiate into ECs and form vascular structures. In this paper, we develop a mathematical model, formulated as a system of nonlinear ordinary differential equations (ODEs), which describes vascular tumour growth with both angiogenesis and vasculogenesis contributing to vessel formation. Submodels describing exclusively angiogenic and exclusively vasculogenic tumours are shown to exhibit similar growth dynamics. In each case, there are three possible scenarios: the tumour remains in an avascular steady state, the tumour evolves to a vascular equilibrium, or unbounded vascular growth occurs. Analysis of the full model reveals that these three behaviours persist when angiogenesis and vasculogenesis act simultaneously. However, when both vascularisation mechanisms are active, the tumour growth rate may increase, causing the tumour to evolve to a larger equilibrium size or to expand uncontrollably. Alternatively, the growth rate may be left unaffected, which occurs if either vascularisation process alone is able to keep pace with the demands of the growing tumour. To clarify further the effects of vasculogenesis, the full model is also used to compare possible treatment strategies, including chemotherapy and antiangiogenic therapies aimed at suppressing vascularisation. This investigation highlights how, dependent on model parameter values, targeting both ECs and EPCs may be necessary in order to effectively reduce tumour vasculature and inhibit tumour growth.     

Effect of Vascular Formed Endothelial Cell Network on the Invasive Capacity of Melanoma Using the In Vitro 3D Co-Culture Patterning Model

In vitro three dimensional (3D) cancer models were developed to observe the invasive capacity of melanoma cell spheroids co-cultured with the vascular-formed endothelial cell network. An array-like multicellular pattern of mouse melanoma cell line B16F1 was developed by magnetic cell labeling using a pin-holder device for allocation of magnetic force. When the B16F1 patterned together with a vascular network of human umbilical vein epithelial cells (HUVEC), spreading and progression were observed along the HUVEC network. The B16F1 cells over 80 µm distance from HUVEC remain in a compact spheroid shape, while B16F1 in the proximity of HUVEC aggressively changed their morphology and migrated. The mRNA expression levels of IL-6, MDR-1 and MMP-9 in B16F1 increased along with the distance the HUVEC network, and these expressions were increased by 5, 3 and 2-fold in the B16F1 close to HUVEC (within 80 µm distance) as compared to that far from HUVEC (over 80 µm distance). Our results clearly show that malignancy of tumor cells is enhanced in proximity to vascular endothelial cells and leads to intravasation.     

Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a fibroblast, adipocyte or endothelial phenotype is dependent on hormonal and extracellular matrix stimulation

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.     

Human endothelial cells cultured on microporous filters used for leukocyte transmigration studies form monolayers on both sides of the filter

Summary A growing number of studies on the mechanism of leukocyte transendothelial migration use endothelial cells grown on microporous filters as an in vitro model of endothelium. Ultrastructural examination of such a model system previously demonstrated that human pulmonary artery endothelial cells (HPAEC) formed confluent monolayers on both sides of the 3-μm-pore filter (Mackarel et al., 1999). To determine whether this was a characteristic specific to pulmonary artery endothelial cells, the growth characteristics of a human pulmonary microvascular endothelial cell type (HMVEC-L) and the widely used human umbilical vein endothelial cells (HUVEC) on 3-μm microporous filters were examined by transmission electron microscopy (TEM). Similar to HPAEC, HMVEC-L and HUVEC were also found to grow on both sides of the filter. All three endothelial cell types were capable of migrating through the 3 μm pores of the filter to form a monolayer on the filter underside. The endothelial cells on the underside were orientated in an inverted position with the luminal surface facing away from the filter. Such ‘bilayer’ formation was observed at a range of seeding densities and in different culture media. Despite the presence of a bilayer of endothelial cells, TEM demonstrated that neutrophils migrated successfully across the cell-filter-cell system. Previous transmigration reports in which an in vitro model similar to ours was used have often assumed only one layer of endothelial cells. The observations reported here indicate that while endothelial cells on microporous filters are useful models for examining leukocyte-endothelial interactions, they are not appropriate for studies examining endothelial cell ‘sidedness.’