Interaction of tumor cells and lymphatic vessels in cancer progression.

Metastatic spread of cancer through the lymphatic system affects hundreds of thousands of patients yearly. Growth of new lymphatic vessels, lymphangiogenesis, is activated in cancer and inflammation, but is largely inactive in normal physiology, and therefore offers therapeutic potential. Key mediators of lymphangiogenesis have been identified in developmental studies. During embryonic development, lymphatic endothelial cells derive from the blood vascular endothelium and differentiate under the guidance of lymphatic-specific regulators, such as the prospero homeobox 1 transcription factor. Vascular endothelial growth factor-C (VEGF-C) and VEGF receptor 3 signaling are essential for the further development of lymphatic vessels and therefore they provide a promising target for inhibition of tumor lymphangiogenesis. Lymphangiogenesis is important for the progression of solid tumors as shown for melanoma and breast cancer. Tumor cells may use chemokine gradients as guidance cues and enter lymphatic vessels through intercellular openings between endothelial cell junctions or, possibly, by inducing larger discontinuities in the endothelial cell layer. Tumor-draining sentinel lymph nodes show enhanced lymphangiogenesis even before cancer metastasis and they may function as a permissive 'lymphovascular niche' for the survival of metastatic cells. Although our current knowledge indicates that the development of anti-lymphangiogenic therapies may be beneficial for the treatment of cancer patients, several open questions remain with regard to the frequency, mechanisms and biological importance of lymphatic metastases.

Quantitative lymphatic vessel trait analysis suggests Vcam1 as candidate modifier gene of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic debilitating disease resulting from a complex interaction of multiple genetic factors with the environment. To identify modifier genes of IBD, we used an F2 intercross of IBD-resistant C57BL/6J-Il10(-/-) mice and IBD-susceptible C3H/HeJBir-Il10(-/-) (C3Bir-Il10(-/-)) mice. We found a prominent involvement of lymphatic vessels in IBD and applied a scoring system to quantify lymphatic vascular changes. Quantitative trait locus (QTL) analyses revealed a large-effect QTL on chromosome 3, mapping to an interval of 43.6 Mbp. This candidate interval was narrowed by fine mapping to 22 Mbp, and candidate genes were analyzed by a systems genetics approach that included quantitative gene expression profiling, search for functional polymorphisms, and haplotype block analysis. We identified vascular adhesion molecule 1 (Vcam1) as a candidate modifier gene in the interleukin 10-deficient mouse model of IBD. Importantly, VCAM1 protein levels were increased in susceptible C3H/HeJ mice, compared with C57BL/6J mice; systemic blockade of VCAM1 in C3Bir-Il10(-/-) mice reduced their inflammatory lymphatic vessel changes. These results indicate that genetically determined expression differences of VCAM1 are associated with susceptibility to colon inflammation, which is accompanied by extensive lymphatic vessel changes. VCAM1 is, therefore, a promising therapeutic target for IBD.

Ultraviolet B irradiation of human skin induces an angiogenic switch that is mediated by upregulation of vascular endothelial growth factor and by downregulation of thrombospondin-1.

BACKGROUND: We have previously demonstrated that skin-specific overexpression of the endogenous angiogenesis inhibitor thrombospondin (TSP)-1 prevented chronic ultraviolet (UV) B-induced angiogenesis, inflammatory cell infiltration and cutaneous photodamage in mice. OBJECTIVES: To elucidate the mechanisms by which acute UVB-induced angiogenesis induces dermal damage, and to study the molecular regulation of acute UVB-induced angiogenesis in human skin. METHODS: We subjected five healthy volunteers to acute UVB irradiation (2 minimal erythema doses) and performed histological analysis at 48 h after UVB irradiation. RESULTS: Histology revealed epidermal hyperplasia, infiltration of elastase-producing neutrophils and elastin fibre damage. Immunohistochemistry for CD31 demonstrated pronounced angiogenesis with a significant increase in both vascular density and vessel size, associated with increased endothelial cell proliferation. Whereas constitutive expression of TSP-1 but only weak expression of vascular endothelial growth factor (VEGF) were detected in normal human epidermis, pronounced downregulation of TSP-1 and upregulation of VEGF were observed in epidermal keratinocytes after acute UVB irradiation. These findings were confirmed by quantitative reverse transcription-polymerase chain reaction analysis after UVB irradiation of cultured HaCaT keratinocytes in vitro. CONCLUSIONS: Together, these data indicate that a disruption of the balance between VEGF and TSP-1 expression leads to a UVB-induced angiogenic switch, facilitating the infiltration of elastase-producing leucocytes and cutaneous photodamage.

The angiogenesis inhibitor vasostatin does not impair wound healing at tumor-inhibiting doses.

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair.

Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.

Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo. Furthermore, VEGF-C induced chemotaxis of macrophages in vitro and in vivo, revealing a potential function of VEGF-C as an immunomodulator. Taken together, our results identify VEGF-C as multifunctional factor involved in regulating tumor lymphangiogenesis, angiogenesis, and immune response.

Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism.

The angiogenic switch during tumorigenesis is thought to be induced by a change in the balance of pro- angiogenic and anti-angiogenic factors. To elucidate the biological role of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, we subjected TSP-2-deficient and wild-type mice to a chemical skin carcinogenesis regimen. Surprisingly, TSP-2 expression was strongly upregulated in the mesenchymal stroma of wild-type mice throughout the consecutive stages of tumorigenesis whereas the angiogenesis factor, vascular endothelial growth factor, was induced predominantly in tumor cells. TSP-2 deficiency dramatically enhanced susceptibility to skin carcinogenesis and resulted in accelerated and increased tumor formation. The angiogenic switch occurred in early stages of pre-malignant tumor formation, and tumor angiogenesis was significantly enhanced in TSP-2-deficient mice. While TSP-2 deficiency did not affect tumor differentiation or proliferation, tumor cell apoptosis was significantly reduced. These results reveal upregulation of an endogenous angiogenesis inhibitor during multi step tumorigenesis and identify enhanced stromal TSP-2 expression as a novel host anti-tumor defense mechanism.

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

Control of hair growth and follicle size by VEGF-mediated angiogenesis.

The murine hair follicle undergoes pronounced cyclic expansion and regression, leading to rapidly changing demands for its vascular support. Our study aimed to quantify the cyclic changes of perifollicular vascularization and to characterize the biological role of VEGF for hair growth, angiogenesis, and follicle cycling. We found a significant increase in perifollicular vascularization during the growth phase (anagen) of the hair cycle, followed by regression of angiogenic blood vessels during the involution (catagen) and the resting (telogen) phase. Perifollicular angiogenesis was temporally and spatially correlated with upregulation of VEGF mRNA expression by follicular keratinocytes of the outer root sheath, but not by dermal papilla cells. Transgenic overexpression of VEGF in outer root sheath keratinocytes of hair follicles strongly induced perifollicular vascularization, resulting in accelerated hair regrowth after depilation and in increased size of hair follicles and hair shafts. Conversely, systemic treatment with a neutralizing anti-VEGF antibody led to hair growth retardation and reduced hair follicle size. No effects of VEGF treatment or VEGF blockade were observed in mouse vibrissa organ cultures, which lack a functional vascular system. These results identify VEGF as a major mediator of hair follicle growth and cycling and provide the first direct evidence that improved follicle vascularization promotes hair growth and increases hair follicle and hair size.

Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4.

The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with beta1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.

In vivo detection of human vascular endothelial growth factor promoter activity in transgenic mouse skin.

We have generated transgenic mice expressing green fluorescent protein (GFP) driven by 2.453-kb (-2,362 to +91) of the 5'-upstream region of the human vascular endothelial growth factor (VEGF) promoter to monitor changes of VEGF gene transcription in situ. Neonatal transgenic mice exhibited GFP-derived fluorescence in tissues that have been previously reported to express VEGF mRNA expression, including lung, cartilage, and brain. In normal skin during postnatal development, moderate fluorescence was observed in the upper epidermis and, more prominently, in the outer root sheath keratinocytes of hair follicles. Strong up-regulation of GFP fluorescence was observed in the hyperplastic epidermis of the wound edge at 48 hours after wounding, whereas little GFP fluorescence was detected in the dermis. In situ hybridization confirmed an identical expression pattern of VEGF mRNA in these wounds. Topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced strong VEGF-GFP expression in suprabasal epidermis. Little or no fibroblast-derived fluorescence was seen both in the wound model and after TPA application. By confocal laser microscopy, increased GFP fluorescence was detectable in the epidermis of intact mouse ear skin as early as 6 hours after topical TPA treatment. Importantly, GFP fluorescence was also measurable in the skin of living transgenic mice. These results resolve the present controversy regarding the ability of VEGF-GFP transgenic mouse models to correctly reflect established patterns of VEGF expression, and show the model to be a powerful tool for the in vivo monitoring of VEGF gene expression.

Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.

Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas.

Inhibition of the vascular endothelial growth factor (VEGF) receptor Flk-1 has been shown to prevent invasion of experimental squamous cell carcinomas (SCC). To directly investigate the role of VEGF in tumor invasion, we stably transfected human SCC-13 cells, which are characterized by a noninvasive phenotype in vivo, with expression vectors containing murine VEGF(164) in sense (SCC/VEGF+) or antisense (SCC/VEGF-) orientation or with vector alone (SCC/vec). SCC/vec cells formed slowly growing, well-differentiated tumors with well-defined borders between tumor and stroma, after intradermal or subcutaneous injection. In contrast, SCC/VEGF+ tumors were characterized by rapid tumor growth, with small cell groups and single cells invading into the surrounding tissue, and by admixture of blood vessels and tumor cells in areas of tumor invasion. We detected an increase in tumor vessel density and size in VEGF-overexpressing tumors, resulting in a more than fourfold increase in total vascular areas. In contrast, SCC/VEGF- clones formed noninvasive, sharply circumscribed tumors with reduced vascular density. These findings demonstrate that selective VEGF overexpression was sufficient to induce tumor invasiveness, and they provide further evidence for an active role of the tumor stroma in cancer progression.

Structure, function, and molecular control of the skin lymphatic system.

The mechanisms of angiogenesis have been studied extensively over the past years. The focus, however, has been almost exclusively on blood vessels, whereas little effort has been directed toward understanding lymphangiogenesis and the role of lymphatic vessels in physiology and pathology. The lymphatic system, acting in concert with the blood vascular system, is of fundamental importance in maintaining tissue homeostasis, and disorders of the lymphatic system are common, often resulting in chronic, disabling conditions. This overview summarizes the most important aspects of the structure and function of the lymphatic system with emphasis on the skin lymphatic vasculature and the differences between blood and lymphatic vessels. Special attention has been given to the methods employed in research of the lymphatic system. Finally, we describe molecular mechanisms involved in the regulation of lymphangiogenesis. Vascular endothelial growth factor and vascular endothelial growth factor-C, expressed by distinct skin cell populations, play an important role in the molecular control of skin angiogenesis and lymphangiogenesis.

Tumor angiogenesis.

In order to grow beyond minimal size and to metastasize, tumors need to induce the growth of new blood vessels (angiogenesis). Whereas in normal tissues, vascular quiescence is maintained by the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli, tumor angiogenesis is induced by increased secretion of angiogenic factors and/or by downregulation of angiogenesis inhibitors. Recent evidence suggests vascular endothelial growth factor (VEGF) as the major tumor angiogenesis factor, promoting tumor growth, invasion, and metastasis. Conversely, blocking of VEGF function inhibits angiogenesis and suppresses tumor growth in vivo. Newly identified members of the VEGF family of angiogenesis factors include placental growth factor, VEGF-B, VEGF-C, and VEGF-D, and show overlapping binding patterns to specific endothelial cell receptors. VEGF-C appears to play a major role as a lymphangiogenesis factor and as a growth factor for Kaposi's sarcoma. In contrast, endogenous inhibitors prevent blood vessel growth in normal tissues. In particular, thrombospondin-1 (TSP-1) and TSP-2 are expressed in normal skin and, when introduced into squamous cell carcinomas, potently inhibit malignant tumor growth via inhibition of tumor angiogenesis.

The role of VEGF and thrombospondins in skin angiogenesis.

The vasculature in adult skin remains normally quiescent, due to the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. However, skin retains the capacity for brisk initiation of angiogenesis, the growth of new blood vessels from preexisting vessels, during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and skin cancers such as cutaneous squamous cell carcinomas. Moreover, cyclic vascular expansion occurs during the growth phase of the hair follicle. Recent evidence suggests vascular endothelial growth factor as the major skin angiogenesis factor. During skin angiogenesis, expression of vascular endothelial growth factor is induced in epidermal keratinocytes by several stimuli including transforming growth factor-alpha and hypoxia, leading to increased vascularization of the dermis. In contrast, vascular endothelial growth factor-C induces skin lymphangiogenesis. Thrombospondin-1 and thrombospondin-2 are endogenous inhibitors of angiogenesis that are expressed in normal skin, maintaining the quiescence of cutaneous vessels. Both inhibitors potently inhibit skin cancer growth via inhibition of tumor angiogenesis. Targeting cutaneous blood vessels represents a promising new therapeutic approach for the treatment of a variety of skin diseases.

Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.

Kaposi's sarcoma is characterized by clusters of spindle-shaped cells that are considered to be tumor cells and by prominent vasculature. Whereas spindle cells are most likely endothelial in origin, it remains controversial whether they are of lymphatic or blood vascular derivation. To test the hypothesis that the lymphangiogenesis factor vascular endothelial growth factor-C and its receptors, KDR and flt-4, are involved in the pathogenesis of Kaposi's sarcoma, we performed in situ hybridizations and immunofluorescent stainings on human immunodeficiency virus-associated Kaposi's sarcoma. Spindle-shaped tumor cells strongly expressed KDR and flt-4 mRNA. Immunofluorescent staining confirmed expression of the flt-4 receptor in Kaposi's sarcoma cells, and double labeling revealed its colocalization with the endothelial cell marker CD31. Vascular endothelial growth factor-C was strongly expressed in blood vessels associated with Kaposi's sarcoma. In vitro, human dermal microvascular endothelial cells also expressed vascular endothelial growth factor-C mRNA that was further upregulated by vascular permeability factor/vascular endothelial growth factor. Vascular endothelial growth factor-C potently stimulated the proliferation of Kaposi's sarcoma tumor cells in vitro. These results demonstrate important paracrine functions of vascular endothelial growth factor-C, produced by blood vessels, in the pathogenesis of cutaneous Kaposi's sarcoma, and suggest a lymphatic origin and/or differentiation of Kaposi's sarcoma tumor cells.

Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas.

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in epithelial tumor development has remained controversial. We studied the in vitro growth characteristics and the in vivo tumor xenograft growth of the human squamous cell carcinoma cell lines A431 and SCC-13, stably transfected to overexpress human TSP-1. Overexpression of TSP-1 inhibited tumor growth of A431 xenotransplants, and completely abolished tumor formation by SCC-13 cells. TSP-1 overexpressing A431 tumors were characterized by extensive areas of necrosis and by decreased tumor vessel number and size. The effects of TSP-1 on tumor cell growth were indirect since tumor cell proliferation rates in vivo and in vitro, anchorage-dependent and -independent growth in vitro, and susceptibility to induction of apoptosis by serum withdrawal were unchanged in TSP-1 overexpressing tumor cells. However, TSP-1 overexpression up-regulated the TSP-1 receptor CD36, leading to enhanced adhesion of A431 cells to TSP-1. These findings establish TSP-1 as a potent inhibitor of angiogenesis and tumor growth in carcinomas of the skin.

Dermatan sulfate activates nuclear factor-kappab and induces endothelial and circulating intercellular adhesion molecule-1.

Proteoglycans (PGs) can influence cell behaviors through binding events mediated by their glycosaminoglycan (GAG) chains. This report demonstrates that chondroitin sulfate B, also known as dermatan sulfate (DS), a major GAG released during the inflammatory phase of wound repair, directly activates cells at the physiologic concentrations of DS found in wounds. Cultured human dermal microvascular endothelial cells exposed to DS responded with rapid nuclear translocation of nuclear factor-kappaB (NF-kappaB), increased expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, and increased ICAM-1 cell surface protein. Heparan sulfate and chondroitin sulfates A and C had no effect on activation of NF-kappaB or induction of ICAM-1. Inhibition of NF-kappaB activation blocked the effect of DS. The increase in cell surface ICAM-1 did not involve TNF-alpha or IL-1 release by endothelial cells, but it was facilitated by autocrine factors whose release was initiated by DS. The ICAM-1-inductive activity of DS was confirmed in vivo. Injection of DS, but not heparin or other chondroitin sulfates, into mice greatly increased circulating levels of soluble ICAM. These data provide evidence that DS, but not other GAGs, initiates a previously unrecognized cell signaling event that can act during the response to injury.

Isolation and culture of microvascular endothelial cells.

The cultivation of endothelial cells from large vessels, predominantly from human umbilical veins (1,2), has become a routine procedure in many laboratories and has contributed to the development of modern vascular biology. However, there is convincing evidence that microvascular endothelial cells display a number of important functional differences, compared to large vessel-derived endothelial cells (3), in particular, with regard to their growth factor response (4,5) and their regulation of adhesion molecule expression (6-8). Since endothelial cells involved in the pathogenesis of tumor angiogenesis, wound healing, and acute and chronic inflammation are predominantly of microvascular origin, techniques have been developed to isolate endothelial cells from small vessels, most frequently from the skin (5,9-13). The culture of human dermal microvascular endothelial cells (HDMEC) has remained problematic because of difficulties in cell isolation, low cell yields, and short lifespans of the isolated cells. In particular, potential contamination of HDMEC cultures with fibroblasts required time-consuming density-gradient centrifugations (5,12) or mechanical removal of fibroblasts (10), and remained problematic after several cell passages. We established a simplified protocol that allows the rapid and reliable immunomagnetic isolation of a well characterized, 100% pure population of HDMEC from neonatal human foreskins. This technique is based on the endothelial cell-specific induction of E-selectin by tumor necrosis factor-alpha (TNF-α) (14), predominantly in postcapillary venule endothelial cells (15), and selection of E-selectin-expressing cells by Dynabeads coupled with an anti-E-selectin monoclonal antibody.