Human dermal microvascular endothelial cells are used to analyze the functions of microvascular endothelium in vitro. However, the low yield and short lifespan of these cells in culture has limited the types of analysis that could be performed. Human microvascular endothelial cells are typically grown in media containing supplements such as dibutyryl cyclic AMP, hydrocortisone, bovine brain extract, and antifungal agents, each of which increase the complexity of experimental design and interpretation of results. In the present study, endothelial cells were transferred after Ulex europeus-I selection into a simplified medium consisting of Endothelial Basal Medium 131, 10% fetal bovine serum, and 2 ng/ml basic fibroblast growth factor and analyzed over 3 mo. The human microvascular endothelial cells expressed the endothelial markers von Willebrand factor, CD31, P-selectin, and E-selectin. In addition, the cells showed increased proliferation in the presence of basic fibroblast growth factor (0.5 ng/ml) or vascular endothelial cell growth factor (10 ng/ml). Tumor necrosis factor-alpha-induced expression of E-selectin was similar in cells at Passages 3, 6, and 12, indicating that the cells maintained responsiveness to cytokines over several weeks. Furthermore, the endothelial cells attained a typical cobblestone morphology with increased cellular density and also formed capillarylike tubes on Matrigel. In summary, the human dermal microvascular endothelial cells display the expected endothelial characteristics when grown for several passages and, therefore, provide a valuable in vitro model for human microvascular endothelium.

Angiostatin, a 38 kilodalton fragment of plasminogen, is a potent inhibitor of angiogenesis. However, little is known about how angiostatin affects endothelial gene expression. To learn more about its effect on endothelial-specific genes implicated in angiogenesis, we examined E-selectin expression and function in bovine capillary endothelial cells treated with recombinant angiostatin. Angiostatin caused a four to five-fold increase in E-selection polypeptide levels in proliferating endothelial cells but little or no increase in confluent cells. P-selection polypeptide levels were unaffected by angiostatin in either proliferating or confluent cells. E-selectin mRNA and adhesion activity in proliferating endothelial cells were also increased by angiostatin. Angiostatin had little effect on the distribution of endothelial cells in G0/G1, S, and G2/M, indicating angiostatin does not alter cell cycle progression significantly. These data demonstrate that angiostatin selectively upregulates E-selectin in proliferating endothelial cells in vitro. This selectivity may provide insights into the mechanism by which angiostatin inhibits tumor growth in vivo without apparent effects on quiescent endothelium.

Angiogenesis plays an important role in breast cancer growth and metastasis. Multiple adhesion molecules have been shown to perform critical functions in the process of angiogenesis. In this study, we analyzed 15 benign and 22 malignant estrogen-receptor-negative and estrogen-receptor-positive breast specimens for the presence of the endothelial cell adhesion molecules E-selectin and P-selectin. We found that E-selectin's expression was increased in the malignant breast tumors compared with their benign counterparts (23.86% of blood vessels versus 2.47%; P = 0.0005). Furthermore, E-selectin staining was found to be significantly increased in the estrogen-receptor-negative carcinomas compared with the estrogen-receptor-positive ones (P = 0.005). In vitro findings strongly correlated with the in vivo findings and showed a higher degree of E-selectin induction in endothelial cells exposed to conditioned media from estrogen-receptor-negative breast cancer cell lines than from estrogen-receptor-positive ones. The degree of E-selectin induction correlated with the amount of interleukin-1 alpha in the tumor-conditioned media. Neutralizing antibodies to interleukin-1 alpha significantly inhibited the E-selectin expression in endothelial cells exposed to tumor-conditioned media. The results indicate that the endothelial E-selectin expression during angiogenesis is related to breast carcinoma progression in vivo and that this component of angiogenesis may be due directly to tumor-cell-secreted interleukin-1 alpha.

OBJECTIVE: E-selectin is an endothelial cell-specific membrane glycoprotein that participates in leukocyte adhesion and has also been suggested to function in angiogenesis. To gain further insights into E-selectin, we analyzed E-selectin polypeptide in proliferating versus quiescent bovine capillary endothelial cells and its expression as a function of the cell cycle. METHODS: E-selectin polypeptide was analyzed by immunoadsorption from 35Scysteine-labeled endothelial cells, by enzyme-linked immunosorbent assay, and by fluorescence-activated cell sorting. The distribution of endothelial cells in Gzero/G1, S, and G2/M phases of the cell cycle was determined using propidium iodide staining of DNA. RESULTS: E-selectin was upregulated in subconfluent proliferating bovine capillary endothelial cells compared to confluent quiescent cultures. The upregulation was independent of activation in that E-selectin was further increased by treatment with tumor necrosis factor alpha or lipopolysaccharide. In contrast to E-selectin, P-selectin and platelet-endothelial cell adhesion molecule-1 did not appear to be regulated by the growth state of the endothelial cells. The distribution of E-selectin-positive cells in GzeroG1, S, and G2/M phases of the cell cycle differed from E-selectin-negative cells in that more of the E-selectin-positive cells were in G2 and M. CONCLUSIONS: Increased E-selectin expression under noninflammatory conditions is correlated with cellular proliferation and G2/M phases of the cell cycle. The expression of E-selectin in proliferating endothelial cells in vitro is consistent with the presence of E-selectin in proliferating endothelial cells in vivo (Kräling et al. [18]).

The levels of E-selectin mRNA and protein were analyzed in bovine capillary cells treated with or without the angiogenesis inhibitor AGM-1470 (also known as TNP-470). Cells treated with AGM-1470 had a two- to sevenfold (median fivefold) increase in E-selectin mRNA compared with little or no increase in P-selectin, PECAM-1 and VCAM-1 mRNA. E-selectin protein was also significantly increased after exposure to AGM-1470. In contrast, there was no detectable effect on PECAM-1 protein. The increase in E-selectin mRNA and protein was always greater with subconfluent growing cells than with confluent cells. This apparent resistance of confluent endothelial cells to AGM-1470 may be relevant to its specificity in vivo. The fact that the effect of AGM-1470 on E-selectin is relatively selective for subconfluent growing cells may provide a clue as to how AGM-1470 is able to both reversibly inhibit endothelial cell proliferation in vitro and inhibit tumor growth in vivo without apparent effects to quiescent endothelium.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

E-selectin, an endothelial-cell-specific leukocyte adhesion molecule, may also function in angiogenesis. To investigate its role in a noninflammatory angiogenic disease, E-selectin was analyzed by immunohistochemistry in specimens of proliferative phase and involutive phase hemangiomas. Hemangioma is an endothelial cell tumor of capillary blood vessels that grows rapidly during infancy and regresses spontaneously during childhood. E-selectin expression was high in proliferative phase specimens and was co-localized with dividing microvascular endothelial cells. Relative to the number of blood vessels, E-selectin declined significantly in involutive phase specimens demonstrating that E-selectin correlates with angiogenesis in the tumors. E-selectin was not detected in quiescent endothelium but was co-localized in dividing microvascular endothelial cells in placenta and neonatal foreskin, two tissues with ongoing growth of microvessels. These in vivo studies support the hypothesis that E-selectin functions in angiogenesis and suggest that E-selectin may be a marker for proliferating endothelium.

Capillaries provide a vast interface between the blood and the tissues that is crucial for regulating nutrient delivery, blood coagulation and transmigration of leukocytes to sites of infection. The growth of new capillaries from pre-existing vessels (angiogenesis) is essential for normal embryogenesis and growth, but also occurs in the development of many diseases. Although relatively little is known about endothelial cell biology, progress is nevertheless being made towards understanding angiogenesis, and several laboratories have begun to identify cell adhesion molecules that may be required for the growth of microvessels.

The levels of P-selectin mRNA and polypeptide were analyzed in bovine capillary cells treated with or without the cytokine tumor necrosis factor-alpha. The 3 kb P-selectin mRNA was upregulated three- to five-fold in cytokine-stimulated cells. The increase in mRNA correlated with a dramatic but short-lived increase in P-selectin polypeptide as determined by metabolic-labeling and immunoadsorption. These data confirm earlier studies on mouse P-selectin expressed in a mouse endothelioma cell line and further indicate that P-selectin function can be regulated not only by rapid translocation to the cell surface but also by cytokine-stimulation of P-selectin biosynthesis.