BCG-induced urinary cytokines inhibit microvascular endothelial cell proliferation.

PURPOSE: Angiogenesis is thought to depend on a net balance of molecules that inhibit or stimulate microvascular endothelial cells. A variety of molecules that affect angiogenesis are induced locally by the administration of intravesical bacille Calmette-Guerin (BCG) for superficial bladder cancer. We sought to determine whether BCG-induced urinary cytokines alter the effects of patient urine on assays of angiogenic activity. MATERIALS AND METHODS: Patients undergoing BCG treatment provided urine samples before and at peak cytokine production times after BCG instillation. Fifty-four urine samples from 8 patients were analyzed by ELISA for a panel of molecules known to affect angiogenesis, and tested for angiogenic activity in human dermal microvascular endothelial cell (HDMEC) proliferation and migration assays. To assess the role of specific BCG-induced cytokines, urinary HDMEC proliferation assays were repeated in the presence of neutralizing antibodies to tumor necrosis factor-alpha (TNF-alpha), interferon-inducible protein-10 (IP-10), and/or interferon-gamma (IFN-gamma). RESULTS: Urinary IFN-gamma, IP-10, TNF-alpha, and vascular endothelial growth factor (VEGF) were induced to nanogram/ml amounts by BCG treatment. While pre-BCG treatment urine samples minimally stimulated microvascular endothelial cell proliferation (+ 9%), post-BCG treatment urine became progressively inhibitory to endothelial cells (to -85%, p = 0.005) during weekly treatment courses. Neutralizing antibodies to TNF-alpha or to IP-10, either alone or in combination, greatly reduced this inhibitory effect. CONCLUSIONS: Intravesical BCG induces a cytokine-rich urinary microenvironment that is inhibitory to human endothelial cells. Urinary cytokine profiles and assays of angiogenic inhibition may provide prognostically important information regarding BCG treatment outcomes.

Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer.

To reveal the antiangiogenic capability of cancer chemotherapy, we developed an alternative antiangiogenic schedule for administration of cyclophosphamide. We show here that this antiangiogenic schedule avoided drug resistance and eradicated Lewis lung carcinoma and L1210 leukemia, an outcome not possible with the conventional schedule. When Lewis lung carcinoma and EMT-6 breast cancer were made drug resistant before therapy, the antiangiogenic schedule suppressed tumor growth 3-fold more effectively than the conventional schedule. When another angiogenesis inhibitor, TNP-470, was added to the antiangiogenic schedule of cyclophosphamide, drug-resistant Lewis lung carcinomas were eradicated. Each dose of the antiangiogenic schedule of cyclophosphamide induced the apoptosis of endothelial cells within tumors, and endothelial cell apoptosis preceded the apoptosis of drug-resistant tumor cells. This antiangiogenic effect was more pronounced in p53-null mice in which the apoptosis of p53-null endothelial cells induced by cyclophosphamide was so vigorous that drug-resistant tumors comprising 4.5% of body weight were eradicated. Thus, by using a dosing schedule of cyclophosphamide that provided more sustained apoptosis of endothelial cells within the vascular bed of a tumor, we show that a chemotherapeutic agent can more effectively control tumor growth in mice, regardless of whether the tumor cells are drug resistant.

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro.

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

Increased apoptosis coincides with onset of involution in infantile hemangioma.

OBJECTIVE: Hemangioma is an endothelial cell tumor that grows rapidly during infancy and regresses slowly during childhood. However, little is known about the natural history of this common tumor. To gain insight into the cellular mechanisms that underlie the switch from uncontrolled growth to involution of endothelium, we investigated the extent of cellular apoptosis versus proliferation in hemangioma specimens that spanned the natural life cycle of the tumor. METHODS: We analyzed apoptosis and cellular proliferation in frozen sections from 16 hemangioma specimens using the TUNEL assay to detect apoptotic cells and the Ki67 antigen to detect dividing cells. RESULTS: Apoptosis was low in proliferative phase hemangiomas but increased fivefold in involutive phase specimens obtained from children one to four years of age. Immunofluorescence double-labeling experiments showed that at least one third of the apoptotic cells were endothelial. As expected, cellular proliferation was high in specimens up to 2 years of age but decreased significantly thereafter. Apoptosis was consistently low in nine normal skin tissues (newborn to 4 years of age) obtained from discarded pathology specimens. CONCLUSIONS: These results suggest that increased apoptosis during the second year of life can offset cellular proliferation and may be involved in initiating regression of hemangioma.

A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors.

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.

Vascular expression of E-selectin is increased in estrogen-receptor-negative breast cancer: a role for tumor-cell-secreted interleukin-1 alpha.

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.

E-selectin is present in proliferating endothelial cells in human hemangiomas.

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.

Transplantation of systemic sclerosis (SSc) skin grafts into severe combined immunodeficient mice: increased presence of SSc leukocytes in SSc skin grafts and autoantibody production following autologous leukocyte transfer.

This study was performed to evaluate the potential of the severe combined immunodeficient (Scid) mouse as a model to study the pathogenesis of systemic sclerosis (SSc). Scid mice were divided into three treatment groups: group 1 received skin grafts and autologous peripheral blood mononuclear cells (PBMC) from either SSc patients or normal individuals, group 2 received only SSc or normal skin grafts, and group 3 received only SSc or normal PBMC transfer. Antinuclear antibodies with a similar expression pattern as in the donor patients were detected in SSc-Scid group 1. Increased numbers of human PBMC were detected after autologous PBMC transfer in normal or SSc skin grafts without crossover into the adjacent mouse tissue. The CD4/CD8 ratio in these infiltrates was approximately 1.0. Although SSc skin transplantations and autologous PBMC transfer into Scid mice did not reproduce the inflammatory events found in the original SSc skin biopsies, these mice could be useful to study the expression of SSc-specific autoantibodies.

Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages.

Systemic sclerosis (SSc) is a generalized autoimmune disorder characterized by immunological abnormalities, microvascular dysfunction, and tissue fibrosis. This study evaluated the inflammatory processes occurring in skin of 7 patients with SSc of recent onset (average disease duration of 10 +/- 3 months) to assess the involvement of monocytes/macrophages during the early stages of SSc. All SSc skin biopsies displayed inflammatory microvascular endothelial cell activation and fibrosis. Increased numbers of infiltrating inflammatory leukocytes were present in affected skin of recent onset SSc (p < 0.01) mainly consisting of CD14-positive monocytes/macrophages (p < 0.02). CD3 T lymphocytes were only slightly elevated in SSc skin (84 +/- 39) compared to normal (51 +/- 12), but the differences were statistically not significant. The ratio of CD14/CD3 cells was substantially higher in affected skin of recent onset SSc (4.0 +/- 2.0) than in normal skin (1.4 +/- 0.5, p < 0.01). Monocytes/macrophages, therefore, are the predominant infiltrating mononuclear cell in skin lesions of recent-onset SSc. These results strongly suggest that CD14-positive monocytes/macrophages play an important role during the early stages of SSc pathogenesis.

Isolation and characterization of microvascular endothelial cells from the adult human dermis and from skin biopsies of patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a rheumatic autoimmune disease without known etiology and pathogenesis. Inflammatory processes with selective microvascular involvement seem to play an important role in the early stages of SSc. EXPERIMENTAL DESIGN: To elucidate the pathogenesis of the selective microvascular involvement in SSc, we have isolated microvascular endothelial cells from the adult human dermis (ADMEC) and for the first time from skin biopsies of patients with SSc (SSc-ADMEC) and established in cell culture. Ulex europaeus I-coated magnetic Dynabeads and the perfusion digestion technique were applied for endothelial cell isolation. RESULTS: The cultured ADMEC and SSc-ADMEC showed the endothelial cell-specific antigenic determinants of intracellular von Willebrand factor and platelet endothelial cell adhesion molecule-1 along their cell-cell borders. These cells displayed specific uptake of acetylated low-density lipoprotein. Normal ADMEC were additionally characterized for angiotensin I-converting enzyme activity. Tumor necrosis factor-alpha activated normal ADMEC and SSc-ADMEC expressed the inflammatory adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, similarly to tumor necrosis factor-alpha-activated large-vessel endothelium represented by human umbilical cord vein endothelial cells. Normal ADMEC and human umbilical cord vein endothelial cells also expressed beta 1- and beta 4-integrin receptors in cell culture. CONCLUSIONS: Normal ADMEC and SSc-ADMEC express endothelial cell-specific antigenic and biochemical determinants in vitro. SSc-ADMEC were for the first time established in cell culture.