Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and migration.

Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.

Polarized expression and basic fibroblast growth factor-induced down-regulation of the alpha 6 beta 4 integrin complex on human microvascular endothelial cells.

Endothelial cells rest on a basement membrane that anchors them to the vessel wall. The alpha 6 beta 4 integrin complex has been described on epithelial cells, frequently localizes to basement-membrane structures, and appears to play a role in binding epithelial cells to laminin. We have determined that human microvascular endothelial cells express the beta 4 integrin chain in vivo and that it preferentially localizes to the endothelial basement membrane. Human microvascular endothelial cells and human umbilical vein endothelial cells also express cell-surface beta 4 in vitro. In addition, the expression of beta 4 appears to be polarized to the undersurface of endothelial cell monolayers in vitro, mimicking its in vivo localization. Stimulation of microvascular endothelial cells with basic fibroblast growth factor or phorbol 12-myristate 13-acetate, agents previously shown to induce endothelial cell migration in vitro, resulted in a marked decrease in cell-surface expression of the beta 4 integrin chain, associated with a decrease in beta 4 mRNA. These data demonstrate that human endothelial cells express the beta 4 integrin chain in vivo and in vitro, the expression of this integrin chain is polarized, and its expression is regulated on microvascular endothelial cells by factors important in wound healing and vascular regeneration.

A 5' portion of the ICAM-1 gene confers tissue-specific differential expression levels and cytokine responsiveness.

Intercellular adhesion molecule-1 (ICAM-1), a cell-adhesion molecule critically involved in leukocyte trafficking and adherence, displays tissue-specific and cytokine-specific expression profiles. Although human dermal microvascular endothelial cells (HDMEC) constitutively express ICAM-1, keratinocytes (HK) do not. Interleukin-1 (IL-1) upregulates ICAM-1 expression in HDMEC, but fails to do so in either HK or A431, a human squamous carcinoma cell line, even though both have IL-1 receptors and express ICAM-1 on exposure to other cytokines. We have previously characterized a human ICAM-1 genomic clone that contains the 5' flanking transcriptional regulatory region. To test the hypothesis that tissue- and cytokine-specific ICAM-1 gene expression results from the interaction of constitutive and inducible tissue-specific trans-acting factors with distinct cis-elements of the ICAM-1 gene, various ICAM-1-based reporter gene (CAT) plasmids were constructed. Transcriptional activity of these various constructs was assessed after transient transfection into HDMEC and A431. A critical ICAM-1 region was identified that conferred enhanced expression of CAT in HDMEC and suppressed expression of CAT in A431. This same region further enhanced CAT expression in transfected HDMEC treated with IL-1 alpha, yet no such enhancement was seen with IL-1 treatment of identically transfected A431. However, treatment of A431 transfectants with IFN gamma did result in enhanced CAT expression, demonstrating reversal of A431 cell context suppression of the ICAM-1-based reporter gene construct. These data implicate the existence of both tissue- and cytokine-specific responsive elements in the 5' flanking region of the ICAM-1 gene and demonstrate that regulatory effects directed by such elements are dependent upon their cellular context. Moreover, they provide the basis for identification of specific cis-acting genetic elements, the trans-acting factors with which they interact, and the molecular mechanisms by which they regulate transcription of the ICAM-1 gene.

Selective upregulation of intercellular adhesion molecule (ICAM-1) by ultraviolet B in human dermal microvascular endothelial cells.

Although a portion of ultraviolet light (UV) penetrates into the dermis, histologic changes that occur within the dermal microvasculature have largely been attributed to the elaboration of biologic substances, such as interleukin 1 (IL-1), from other constitutive cells of the skin, as opposed to a direct effect of UV on the endothelial cell. As a potential model for understanding early molecular events occurring in UV-induced cutaneous inflammation, we have examined the direct effects of UVB, as well as cytokine-positive controls, upon human dermal microvascular endothelial cells (HDMEC) cell adhesion molecule (CAM) gene expression. Cultured HDMEC were exposed to varying dosages of UVB, and examined for cell surface and mRNA expression of the CAMs intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (formerly ELAM-1). Following UVB exposure, dose-dependent increases in baseline cell surface expression of ICAM-1 were demonstrated by fluorescence-activated cell sorter analysis with concomitant increases in ICAM-1 mRNA, as shown by Northern blot analysis; there was no induction of either E-selectin or VCAM-1. The UVB-induced ICAM-1 upregulation could not be blocked by antibodies to IL-1 or tumor necrosis factor alpha (TNF-alpha). In fact, ICAM-1 gene regulatory region based CAT reporter gene plasmids, including constructs containing IL-1- and TNF-alpha-responsive elements, did not display increased CAT expression after transfection into HDMEC followed by UVB exposure, though control cytokine-treated transfectants did. Thus, UVB selectively upregulates ICAM-1, but not E-selectin or VCAM-1, mRNA and cell surface expression in HDMEC, and this upregulation is not dependent upon the autologous secretion and activity of either IL-1 or TNF-alpha.

Nonsteroidal progesterone receptor ligands. 2. High-affinity ligands with selectivity for bone cell progesterone receptors.

A novel series of nonsteroidal heterocycles was discovered which display cell-type selective, high-affinity (nanomolar) binding to the progesterone receptors from TE85 osteosarcoma cells but > 1 microM binding affinity to the progesterone receptors from T47D and ZR75 human breast carcinoma cells. Structure-activity relationships were developed for a set of these compounds, and a representative analog 1-(3,4-dichlorobenzoyl)-3-phenyl-1,4,5,6-tetrahydropyridazine++ + (1i, RWJ 25333) was chosen for further evaluation. RWJ 25333 stimulated the in vitro proliferation of human osteoblast-like cells but not human breast cells.

Busy physicians and preventive services for adults.

OBJECTIVE: To study the relationship between overall productivity and the rates at which primary care physicians, in a fee-for-service setting, deliver or prescribe preventive services to adult patients. PATIENTS AND METHODS: The charts of 452 adult patients treated by 8 family practitioners and 5 internists in a fee-for-service practice setting were randomly selected and abstracted for provision of 10 preventive services over a 27-month period. The percentage of eligible patients screened for each service was correlated with the production of each physician measured in relative value units (RVUs). RESULTS: The correlation coefficient between RVUs and the aggregate of the 10 services was 0.23 (95% confidence interval [CI], -0.36 to 0.70). The individual correlation coefficients between RVUs and 9 of the 10 preventive services ranged from -0.05 to 0.43. For cervical cancer screening, however, the correlation coefficient was -0.72 (95% CI, -0.91 to -0.24). CONCLUSION: With the exception of screening for cervical cancer, the data presented in this study do little to support physicians' common belief that lack of time is the reason they are unable to incorporate prevention strategies into their clinical practice.

Matrix metalloproteinases: pro- and anti-angiogenic activities.

Matrix metalloproteinases (MMP) are a family of structurally related proteinases most widely recognized for their ability to degrade extracellular matrix, although recent investigations have demonstrated other biologic functions for these enzymes. MMP are typically not constitutively expressed, but are regulated by: (1) cytokines, growth factors, and cell-cell and cell-matrix interactions that control gene expression; (2) activation of their proenzyme form; and (3) the presence of MMP inhibitors [tissue inhibitors of metalloproteinases, (TIMP)]. MMP have important roles in normal processes including development, wound healing, mammary gland, and uterine involution, but are also involved in angiogenesis, tumor growth, and metastasis. Angiogenesis, characteristically defined as the establishment of new vessels from pre-existing vasculature, is required for biologic processes such as wound healing and pathologic processes such as arthritis, tumor growth, and metastasis. Blocking of MMP activity has been studied for potential therapeutic efficacy in controlling such pathologic processes. Synthetic MMP inhibitors, most notably the hydroxymates, have been engineered for this purpose and are presently in clinical trial. These inhibitors may have broad versus specific MMP inhibitory activity. As increased non-matrix degrading capabilities of MMP are recognized, however, i.e., cytokine activation, processing of proteins to molecules of distinct biologic function, it becomes less clear whether the nonselective inhibition of MMP activity for all pathologic processes involving MMP is appropriate. This review focuses upon the contribution of MMP to the process of tumor invasion and angiogenesis, and discusses the design and use of MMP inhibitors as therapeutic agents in these processes.

Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases.

The serine proteinases plasmin and thrombin convert proenzyme matrix metalloproteinases (MMPs) into catalytically active forms. In addition, we demonstrate that plasmin(ogen) and thrombin induce a significant increase in secretion of activated murine macrophage elastase (MMP-12) protein. Active serine protease is responsible for induction, as demonstrated by the absence of MMP-12 induction in plasminogen(Plg)-treated urokinase-type plasminogen activator-deficient macrophages. Since increased MMP-12 protein secretion was not accompanied by an increase in MMP-12 mRNA, we examined post-translational mechanisms. Protein synthesis was not required for early release of MMP-12 but was required for later secretion of activated enzyme. Immunofluorescent microscopy demonstrated basal expression in macrophages that increased following serine proteinase exposure. Inhibition of MMP-12 secretion by hirudin and pertussis toxin demonstrated a role for the thrombin G protein-coupled receptor (protease-activated receptor 1 (PAR-1)). PAR-1-activating peptides were able to induce MMP-12 release. Investigation of signal transduction pathways involved in this response demonstrate the requirement for protein kinase C, but not tyrosine kinase, activity. These data demonstrate that plasmin and thrombin regulate MMP-12 activity through distinct mechanisms: post-translational secretion of preformed MMP-12 protein, induction of protein secretion that is protein kinase C-mediated, and extracellular enzyme activation. Most importantly, we show that serine proteinase MMP-12 regulation in macrophages occurs via the protein kinase C-activating G protein-coupled receptor PAR-1.

Thrombospondin-1, a natural inhibitor of angiogenesis, is present in vitreous and aqueous humor and is modulated by hyperglycemia.

Negative regulators of angiogenesis play a major role in maintaining vascular homeostasis. Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. This report examines the presence of TSP1 in ocular samples and determines whether its production is altered in diabetes. Western blot analysis detected a 140 kDa antiangiogenic fragment of TSP1(gp140) in vitreous samples prepared from normal human and rat eyes. Intact TSP1 was detected in aqueous humor samples prepared from normal rat and bovine eyes. In contrast, TSP1 was virtually absent in vitreous and aqueous humor samples prepared from diabetic rat eyes. Furthermore, production of TSP1 by microvascular endothelial cells in culture was sensitive to high concentrations of glucose. Retinal blood vessels appeared nonuniform and dilated in diabetic animals when compared to control animals. These results demonstrate that TSP1 and its antiangiogenic fragment are present in aqueous humor and vitreous of normal rat eyes and are dramatically reduced in diabetes. Thus, TSP1 may play a role in ocular vascular homeostasis and its absence may contribute to vascular dysfunctions associated with diabetes.

Matrix metalloproteinases generate angiostatin: effects on neovascularization.

Angiostatin, a cleavage product of plasminogen, has been shown to inhibit endothelial cell proliferation and metastatic tumor cell growth. Recently, the production of angiostatin has been correlated with tumor-associated macrophage production of elastolytic metalloproteinases in a murine model of Lewis lung cell carcinoma. In this report we demonstrate that purified murine and human matrix metalloproteinases generate biologically functional angiostatin from plasminogen. Macrophage elastase (MMP-12 or MME) proved to be the most efficient angiostatin-producing MMP. MME was followed by gelatinases and then the stomelysins in catalytic efficiency; interstitial collagenases had little capacity to generate angiostatin. Both recombinant angiostatin and angiostatin generated from recombinant MME-treated plasminogen inhibited human microvascular endothelial cell proliferation and differentiation in vitro. Finally, employing macrophages isolated from MME-deficient mice and their wild-type littermates, we demonstrate that MME is required for the generation of angiostatin that inhibits the proliferation of human microvascular endothelial cells.