Contribution of outgrowth endothelial cells from human peripheral blood on in vivo vascularization of bone tissue engineered constructs based on starch polycaprolactone scaffolds

In the present study we assessed the potential of human outgrowth endothelial cells (OEC), a subpopulation within endothelial progenitor cell cultures, to support the vascularization of a complex tissue engineered construct for bone. OEC cultured on starch polycaprolactone fiber meshes (SPCL) in monoculture retained their endothelial functionality and responded to angiogenic stimulation by VEGF (vascular endothelial growth factor) in fibrin gel-assays in vitro. Co-culture of OEC with human primary osteoblasts (pOB) on SPCL, induced an angiogenic activation of OEC towards microvessel-like structures achieved without additional supplementation with angiogenic growth factors. Effects of co-cultures with pOB on the vascularization process by OEC in vivo were tested by subcutaneous implantation of Matrigel plugs containing both, OEC and pOB, and resulted in OEC-derived blood vessels integrated into the host tissue and anastomosed to the vascular supply. In addition, morphometric analysis of the vascularization process by OEC indicated a better performance of OEC in the co-cultures with primary osteoblasts compared to monocultures of OEC. The contribution of OEC to vascular structures and the beneficial effect of the co-culture with primary human osteoblasts on the vascularization in vivo was additionally proven by subcutaneous implantation of pre-cellularized and pre-cultured SPCL constructs. OEC contributed to the vascular structures, by generating autogenic vessels or by incorporation into chimeric vessels consisting of both, human and mouse endothelial cells. The current data highlight the vasculogenic potential of OEC for bone tissue engineering applications and indicate a beneficial influence of constructs including both osteoblasts and endothelial cells for vascularization strategies.     

Modulation of in vitro angiogenesis in a three-dimensional spheroidal coculture model for bone tissue engineering

One of the major challenges in tissue engineering of bone substitutes remains vascularization of the transplant. We have developed a three-dimensional collagen-based coculture system to assess interactions between human endothelial cells (hECs) and human osteoblasts (hOBs) in vitro. Human umbilical vein endothelial cells (HUVECs) were grown as three-dimensional multicellular spheroids and seeded in a collagen matrix to assess sprouting of the spheroids, that is, formation of tubelike structures resembling early capillaries. Direct cell contact between hOBs and HUVECs was established by incorporating hOBs into the EC spheroids, thus forming heterogeneous cospheroids. Spatial organization of cospheroids and sprout configuration were assessed by immunohistochemical wholemount staining techniques and confocal laser microscopy. Cumulative sprout length of spheroids was quantitatively analyzed by digital imaging planimetry. In this model HUVECs and hOBs formed heterogeneous cospheroids with distinct spatial organization. The ability of HUVEC spheroids to form tubelike structures on angiogenic stimulation with vascular endothelial growth factor and basic fibroblast growth factor was suppressed in heterogeneous HUVEC/hOB cospheroids. The model system introduced in this study may be useful to assess the mechanisms involved in regulating angiogenesis during bone formation and to further investigate the mechanisms by which heterotypic cell-cell interactions inhibit endothelial tube formation for applications in bone tissue engineering.     

Microvessel-like structures from outgrowth endothelial cells from human peripheral blood in 2-dimensional and 3-dimensional co-cultures with osteoblastic lineage cells

Tissue regeneration involves complex processes in the interaction between different cell types that control the process of neo-vascularization. In bone, osteoblasts and bone marrow stem cells provide cue elements for the proliferation of endothelial cells, differentiation of endothelial precursors, and the maturation of a vascular network. In this study, we investigated outgrowth endothelial cells (OECs), a potential source of autologous endothelial cells derived from human peripheral blood, in direct 2-dimensional (2-D) and 3-D co-culture systems with cells relevant for the regeneration of bone tissue, such as osteoblasts. In the co-cultures, OECs were evaluated in terms of their stability as an endothelial population at the single cell level using flow cytometry and their ability to establish a pre-vascular network at the light-microscopical and ultra-structural level. In co-cultures with the osteoblast cell line MG63 and with human primary osteoblasts (pOBs), OECs, in contrast to human umbilical vein endothelial cells, formed highly organized microvessel-like structures. These microvessel-like structures included the formation of a vascular lumen with tight junctional complexes at intercellular contacts of endothelial cells. In the co-culture, the formation of this vascular network was achieved in the standard growth medium for OECs. Furthermore, using a rotating culture vessel system, 3-D co-cultures consisting of OECs and pOBs were generated. Based on these observations, we conclude that OECs could provide a valuable source of autologous endothelial cells for the generation of complex tissue-engineered tissues.     

Co-culture of bone marrow fibroblasts and endothelial cells on modified polycaprolactone substrates for enhanced potentials in bone tissue engineering

The creation of a vascularized bed makes the survival of seeded cells on 3-dimensional scaffolds much more likely. However, relying purely on random capillary ingrowth into the porous scaffolds from the host may compromise vascularization of a scaffold. One solution is to transplant cells capable of differentiating into new blood vessels into the scaffolds to accelerate the creation of a vascularized scaffold. Because endothelial cells are the key cells involved in blood vessel formation, the present study was designed to investigate the development of a biomaterial surface that supports endothelial cell attachment and proliferation. The subsequent effects of the material surface modifications on the differentiation and proliferation of human bone marrow-derived fibroblasts (HBMFs) when grown in co-culture with a human bone marrow endothelial cell line (HBMEC-60) were studied. Endothelialization studies showed that the gelatin-coated and hydroxyapatite-coated substrates were superior for HBMEC-60 attachment and proliferation to hydrolyzed-only or untreated polycaprolactone substrates. Co-culture studies showed that the presence of the HBMEC-60 specifically enhanced HBMF cell proliferation and differentiation and that this effect was not observed with co-culture with skin fibroblasts. It is concluded that the co-culture of endothelial cells with HBMFs could be a promising culture system for bone tissue- engineering applications.     

Influence of polymer content in Ca-deficient hydroxyapatite–polycaprolactone nanocomposites on the formation of microvessel-like structures

Calcium phosphate (CaP) ceramics are widely used in bone tissue engineering due to their good osteoconductivity. The mechanical properties of CaP can be modified by the addition of small volume fractions of biodegradable polymers such as polycaprolactone (PCL). Nevertheless, it is also important to evaluate how the polymer content influences cell–material or cell–cell interactions because of potential consequences for bone regeneration and vascularization. In this study we assessed the general biocompatibilty of Ca-deficient hydroxyapatite (CDHA)–PCL disks containing nominally 11 and 24% polycaprolactone using human umbilical vein endothelial cells and human primary osteoblasts. Confocal microscopy showed that both CDHA–PCL variants supported the growth of both cell types. In terms of the endothelial cells grown on CDHA–PCL nanocomposites with 24% PCL, an increased expression of the endothelial marker vWF compared to CDHA–PCL with 11% PCL was observed in real-time polymerase chain reaction analysis. In addition to monocultures, co-cultures of outgrowth endothelial cells, derived from peripheral blood, and primary osteoblasts were assessed as an example of a more complex test system for bone regeneration and vascularization. Constructs based on CDHA with different PCL contents were investigated with regard to the formation of microvessel-like structures induced by the co-culture process using confocal microscopy and quantitative image analysis. Furthermore, the osteogenic differentiation of the co-culture was assessed. As a result, more pre-vascular structures were observed after 1 week on the CDHA–PCL disks with 24% PCL, whereas after 4 weeks of culture the extent of microvessel-like structure formation was slightly higher on the CDHA with 11% PCL. In contrast to this, variation of PCL content had no effect on the osteogenic differentiation in the co-culture.     

Oxygen-Tension Controlled Matrices for Enhanced Osteogenic Cell Survival and Performance

The success of a clinically-applicable bone tissue engineering construct for large area bone defects depends on its ability to allow for homogeneous bone regeneration throughout the construct. Insufficient vascularization, and consequently inadequate oxygen tension, throughout constructs has been largely cited as the most significant obstacle facing successful bone regeneration in large area defects. The development of constructs that support bone and vessel-forming cell growth and function throughout the scaffold structure are desired for large-area bone defect repair. Here, we developed oxygen tension-controlled matrices that support more homogenous oxygen levels throughout the constructs. Specifically, we examined polylactic co-glycolic acid (PLGA) scaffolds with optimized pore distribution and the percent pore volumes, and demonstrated significantly decreased oxygen and pH gradient from the exterior of the construct to the interior after long-term cell culture in vitro. We confirmed the ability of these optimized constructs to support the cellular survival via live/dead assay. In addition, we examined their ability to support the maintenance of two clinically relevant progenitor cell populations for bone tissue engineering and vascularization, namely mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs), and confirmed the expression of key bone and vascular markers via immunofluorescence.     

Fibronectin coating of collagen modules increases in vivo HUVEC survival and vessel formation in SCID mice

Modular tissue engineering is a novel approach to creating scalable, self-assembling, three-dimensional tissue constructs with inherent vascularization. Under initial methods, the subcutaneous implantation of human umbilical vein endothelial cell (HUVEC)-covered collagen modules in immunocompromised mice resulted in significant host inflammation and limited HUVEC survival. A minimally invasive injection technique was used to minimize surgery-related inflammation, and cell death was attributed to extensive apoptosis within 72 h of implantation. Coating collagen modules with fibronectin (Fn) was shown in vivo to reduce short-term HUVEC TUNEL staining by nearly 40%, while increasing long-term HUVEC survival by 30-45%, relative to collagen modules without fibronectin. Consequently, a ～100% increase in the number of HUVEC-lined vessels was observed with Fn-coated modules, as compared to collagen-only modules, at 7 and 14 days post-implantation. Furthermore, vessels appeared to be perfused with host erythrocytes by day 7, and vessel maturation and stabilization was evident by day 14.     

Platelet lysate coating on scaffolds directly and indirectly enhances cell migration,improving bone and blood vessel formation

Suitable colonization and vascularization of tissue-engineered constructs after transplantation represent critical steps for the success of bone repair. Human platelet lysate (hPL) is composed of numerous growth factors known for their proliferative, differentiative and chemo-attractant effects on various cells involved in wound healing and bone growth. The aim of this study was to determine whether the delivery of human mesenchymal stromal cells (hMSC) seeded on hPL-coated hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) scaffolds could enhance vascularization and bone formation, as well as to investigate the mechanisms by which hMSC participate in tissue regeneration. Our study demonstrates that hPL can be coated on HA/β-TCP scaffolds, which play direct and indirect effects on implanted and/or resident stem cells. Effectively, we show that hPL coating directly increases chemo-attraction to and adhesion of hMSC and endothelial cells on the scaffold. Moreover, we show that hPL coating induces hMSC to produce and secrete pro-angiogenic proteins (placental growth factor and vascular endothelial growth factor) which allow the proliferation and specific chemo-attraction of endothelial cells in vitro, thus improving in vivo neovascularization and new bone formation. This study highlights the potential of functionalizing biomaterials with hPL and shows that this growth factor combination can have synergistic effects leading to enhanced bone and blood vessel formation.     

Response of micro- and macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering

The establishment of a functional vasculature is as yet an unrealized milestone in bone reconstruction therapy. For this study, fiber-mesh scaffolds obtained from a blend of starch and poly(caprolactone) (SPCL), that have previously been shown to be an excellent material for the proliferation and differentiation of bone marrow cells and thereby represent great potential as constructs for bone regeneration, were examined for endothelial cell (EC) compatibility. To be successfully applied in vivo, this tissue engineered construct should also be able to support the growth of ECs in order to facilitate vascularization and therefore assure the viability of the construct upon implantation. The main goal of this study was to examine the interactions between ECs and SPCL fiber meshes. Primary cultures of HUVEC cells were selected as a model of macrovascular cells and the cell line HPMEC-ST1.6R as a model for microvascular ECs. Both macro- and microvascular ECs adhered to SPCL fiber-mesh scaffolds and grew to cover much of the available surface area of the scaffold. In addition, ECs growing on the SPCL fibers exhibited a typical morphology, maintained important functional properties, such as the expression of the intercellular junction proteins, PECAM-1 and VE-cadherin, the expression of the most typical endothelial marker vWF and sensitivity to pro-inflammatory stimuli, as shown by induction of the expression of cell adhesion molecules (CAMs) by lipopolysaccharide (LPS). These data indicate that ECs growing on SPCL fiber-mesh scaffolds maintain a normal expression of EC-specific genes/proteins, indicating a cell compatibility and potential suitability of these scaffolds for the vascularization process in bone tissue engineering in vivo.     

Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration

All engineered bioartificial structures developed for tissue regeneration require oxygen and nutrients to establish proper physiological functions. Aiming to improve vascularization during dermal regeneration, we combined the use of a bioartificial collagen scaffold and a defined human mesenchymal cell (MC) line. This cell line, termed V54/2, exhibits typical morphologic and immunohistochemical characteristics of MC. V54/2 cells seeded in the scaffold were able to survive, proliferate, and secrete significant amounts of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) during 2 weeks in vitro. To induce dermal regeneration, scaffolds with or without cells were transplanted in a nude mice full skin defect model. After 2 weeks of transplantation, scaffolds seeded with V54/2 cells showed more vascularization during the dermal regeneration process than controls, and the presence of human cells in the regenerating tissue was detected by immunohistochemistry. To confirm if local presence of angiogenic growth factors is sufficient to induce neovascularization, scaffolds were loaded with VEGF and bFGF and used to induce dermal regeneration in vivo. Results showed that scaffolds supplemented with growth factors were significantly more vascularized than control scaffolds (scaffolds without growth factors). The present work suggests that combined use of MC and bioartificial scaffolds induces therapeutic angiogenesis during the scaffold-based dermal regeneration process.     

血管内皮细胞促进组织工程骨血管化的研究进展

组织工程骨的血管化问题严重制约了大块组织工程骨的发展和运用.血管内皮细胞对组织工程骨血管化具有重要促进作用,根据血管化过程中血管内皮细胞的来源不同,将血管化过程分血管发生和血管生成两种形式.成骨细胞和血管内皮细胞共同培养时可以相互促进生长.目前对组织工程骨血管化进行了血管内皮细胞新的来源,细胞混合培养和体内试验等方面的探索.     

A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.     

Endothelial Cell–Smooth Muscle Cell Co-Culture in a Perfusion Bioreactor System

Vascular endothelial cells (EC) are exposed to a complex biomechanical environment in vivo and are responsible for relaying important messages to the underlying tissue. EC and smooth muscle cells (SMC) communicate to regulate vascular development and function. In this work, a vascular perfusion bioreactor is used to grow tubular constructs seeded with EC and SMC under pulsatile shear stress in long-term co-culture to study the effects of EC on SMC function. SMC seeded into porous poly(glycolic acid) tubular scaffolds are cultured in the bioreactor for 25 days. Constructs are seeded with EC on day 10 or day 23 creating 2-day (short-term) or 15-day (long-term) EC and SMC co-cultures. Long-term EC–SMC co-culture significantly increases cell proliferation and downregulates collagen and proteoglycan deposition compared to short-term co-culture. After 25 days of culture, 15-day co-culture constructs have a more uniform cell distribution across the construct thickness and SMC express a more contractile phenotype compared to 2-day co-culture constructs. These data demonstrate strong interactions between SMC and EC in the bioreactor under physiologically relevant conditions. Thus, the vascular construct perfusion bioreactor is an important tool to investigate cell–cell and cell–extracellular matrix interactions in vascular cell biology and tissue engineering.     

Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes

Open-cell hollow fibers made of polyethersulfone (PES) manufactured in the absence of solvents with pore diameters smaller than 100 microm were examined for vascularization by human endothelial cells. The goal of this study was to determine whether the 3-D porous character of the PES surface affected human endothelial cell morphology and functions. Freshly isolated human endothelial cells from the skin (HDMEC), from the lung (HPMEC) and from umbilical cords (HUVEC) and two human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS.c1 were added to PES fibers and cell adherence and growth was followed by confocal laser scanning microscopy. Prior coating of PES with gelatin or fibronectin was necessary for adhesion and spreading of cells over the uneven porous surface with time. Confluent cells exhibited typical strong PECAM-1 expression at cell-cell borders. Little expression of the activation markers E-selectin, ICAM-1, and VCAM-1 was observed by RT-PCR of endothelial cells growing on PES. However, after stimulation for 4h by LPS, activation of these markers was observed and it was shown by immunofluorescent staining that induction occurred in most of the cells, thus confirming an intact functionality. Finally, cells growing as a monolayer on PES migrated to form microvessel-like structures when placed under conditions that stimulated angiogenesis. Thus, human endothelial cells grown on fibronectin-coated PES fibers retain important endothelial-cell specific morphological and functional properties and PES may serve as a useful biomaterial in tissue engineering and biotechnology applications.     

Survival of endothelial cells in vitro on Paclitaxel-loaded coronary stents

Coronary stents that are developed for use with balloon angioplasty are known to cause acute occlusion and long-term stenosis. It is likely that a controlled release of drugs at the site of stent implantation might inhibit the proliferation of vascular smooth muscle cells (VSMC) and reduce restenosis. However, if the drug is necrotic and affects cell survival near the implant, it may interrupt the local tissue regeneration. Different methods have been used for the immobilization of drugs with stents to get an effective concentration that inhibits cell proliferation. The objective of this study is to assess the effectiveness of Paclitaxel-loaded stents by immobilization with a biodegradable polymer, to inhibit cell proliferation. The cells used for the evaluation are human umbilical vein endothelial cells (HUVEC) and the proliferation rate of these cells on the drug-coated stent is compared against an uncoated stent for a 72-h period. Evaluations were also made to differentiate between cell apoptosis and necrosis to prove that the drug released is not deleterious to the surrounding tissue.While a similar initial cell adhesion is observed in bare and coated stents, the proliferation of HUVEC is negligible when grown on a drug-coated stent (p < 0.001). By specific staining techniques, the cells on the drug-coated stents are found to be apoptotic and not necrotic, throughout the evaluation period. In vitro leukocyte adhesion and platelet deposition on the drug-coated stents are found to be low when they are exposed to human blood and platelet-rich plasma (PRP), suggesting that the coated stents may not be thrombogenic in vivo. Therefore, drug coating of stents using the described technique may have a considerable promise for the prevention of neointimal proliferation, restenosis, and associated failure of angioplasty.     

In-vitro inhibitory effect of EGFL7-RNAi on endothelial angiogenesis in glioma

Objective: To investigate the role and mechanism of epidermal growth factor like domain 7 (EGFL7) in glioma angiogenesis by cell co-culture and RNA interference. Methods: NSCs-HUVECs co-culture system was established using Transwell culturing techniques. The interactions between glioma and endothelial cells were simulated in-vitro. Cellular expression of EGFL7 in NSCs and HUVEC was targeted and suppressed by lentiviral vector carrying siRNA. The effect of EGFL7 on angiogenesis in glioma in-vitro micro-environment was detected by endothelial cell proliferation, adhesion and tube formation assay. Results: Following EGFL7 gene silencing, expression of EGFL7 in HUVECs was reduced and cell adhesion capability was inhibited significantly. Endothelial cells failed to form a lumen-like structure after EGFL7 gene silencing, shown by the tube formation assay. Conclusion: By regulating endothelial cell adhesion, EGFL7 plays a key role in the regulation of glioma angiogenesis.     

Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration

Recent study showed that mesenchymal stem cells (MSC) could inhibit apoptosis of endothelial cells in hypoxic condition, increase their survival, and stimulate the angiogenesis process. In this project we applied Laser-Induced-Forward-Transfer (LIFT) cell printing technique and prepared a cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSC (hMSC) in a defined pattern for cardiac regeneration. We seeded HUVEC and hMSC in a defined pattern on a Polyester urethane urea (PEUU) cardiac patch. On control patches an equal amount of cells was randomly seeded without LIFT. Patches were cultivated in?vitro or transplanted in?vivo to the infarcted zone of rat hearts after LAD-ligation. Cardiac performance was measured by left ventricular catheterization 8 weeks post infarction. Thereafter hearts were perfused with fluorescein tomato lectin for the assessment of functional blood vessels and stored for histology analyses. We demonstrated that LIFT-derived cell seeding pattern definitely modified growth characteristics of co-cultured HUVEC and hMSC leading to increased vessel formation and found significant functional improvement of infarcted hearts following transplantation of a LIFT-tissue engineered cardiac patch. Further, we could show enhanced capillary density and integration of human cells into the functionally connected vessels of murine vascular system. LIFT-based Tissue Engineering of cardiac patches for the treatment of myocardial infarction might improve wound healing and functional preservation.     

In vitro reconstruction of human dermal equivalent enriched with endothelial cells

Experiences coming from many cell-culture studies has brought about the concept that tissue and organ reconstruction should be performed in a three-dimensional environment as it normally occurs in vivo. As far as endothelial cell culture is concerned, it has been shown that angiogenesis can be successfully achieved only when cells are cultured in the presence of collagen-based matrices or basal membrane substrates. The aim of the present investigation is to demonstrate that human umbilical vein endothelial cells (HUVEC) can be grown and differentiated on an artificial dermis obtained by fibroblasts cultured on hyaluronic acid-based scaffolds. For this purpose, we have cultured HUVEC, retrieved by collagenase digestion of perfused human umbilical vein either alone and with fibroblast at 1/1 ratio into HYAFF-11 non-woven mesh. Cultures were maintained for up to 3 weeks. Samples were taken at different time points within this period for the MTT proliferation test and for immunohistochemical analysis. Our results demonstrate that hyaluronan-based biomaterials (HYAFF-11 NW mesh) represent a suitable substrate for HUVEC adhesion, proliferation and reorganization in microcapillary network.     

Influence of angiogenesis inhibitors on endothelial cell morphology in vitro

Human umbilical vein endothelial cells (HUVEC) propagated in co-culture with fibroblasts form capillary-like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co-cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co-culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary-like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co-culture untreated HUVEC formed capillary-like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel-Palade bodies and transport vesicles. Immuno-EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell-cell contacts, and at the luminal and abluminal side of the capillary-like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor-treated co-cultures. Our results demonstrate that treatment with levamisole or anti-VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP-470 results in formation of short endothelial cell cords. We discuss the implications of these findings.     

Investigation of interleukin 2 receptors on human endothelial cells.

We have demonstrated that both recombinant and purified IL-2 exert a direct effect on quiescent human microvascular endothelial cells in vitro, causing the cells to enter the cell cycle and proliferate (Hicks et al., 1989). In this study we have identified IL-2 receptors (R) on both human umbilical vein (HUVEC) and neonatal foreskin (HCEC) endothelial cells. The techniques used to identify the receptors included proliferation studies, flow cytometry and immunofluorescence. Results indicate that both HUVEC and HCEC possess low numbers of receptors since both cell types proliferate in response to IL-2. The number of receptors on the cell surface vary according to passage number and culture conditions. Immunofluorescent studies show discrete areas of staining on the cell membrane. These combined results suggest that human vascular endothelial cells possess IL-2R.