Abstract. In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures, bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFet induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFa were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFot, suggesting that TNFet-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. e-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFoL or u-PA did not induce tubular structures without additional growth factors, bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFot itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFa, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFet is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.

Matrix metalloproteinases (MMPs) play a role in tissue remodelling and angiogenesis. We have investigated the expression and regulation of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin), MMP-9 (gelatinase B) and their inhibitors TIMP-1 and TIMP-2 in human umbilical vein, femoral vein and microvascular endothelial cells, and compared these data with those obtained with human synovial fibroblasts. Non-stimulated vein endothelial cells expressed the mRNAs for MMP-1, MMP-2, TIMP-1 and TIMP-2. MMP-3 mRNA and protein were undetectable or only weakly expressed, but could be stimulated by the inflammatory mediator tumour necrosis factor alpha (TNF alpha). The expression of MMP-3 and MMP-1 was further enhanced by phorbol 12-myristate 13-acetate (PMA). Phorbol ester also induced TIMP-1 and MMP-9, the expression of the latter being further enhanced by TNF alpha or interleukin 1 alpha (IL-1 alpha). Similar stimulatory effects were observed in microvascular endothelial cells. Hence the inflammatory mediator TNF alpha induces/enhances the production of several matrix metalloproteinases in human endothelial cells. On the other hand, MMP-2 and TIMP-2 were not affected or were affected in a variable way by TNF alpha and/or phorbol ester, suggesting a dissimilar regulation of these proteins. The cyclic AMP-enhancing agent forskolin affected the production of MMPs in a cell-type-specific way. In human vein endothelial cells it enhanced the PMA-mediated induction of MMP-9, whereas it suppressed this induction in human microvascular endothelial cells and in synovial fibroblasts. On the other hand, forskolin suppressed the PMA-mediated induction of MMP-1 and MMP-3 in synovial fibroblasts, while it enhanced or did not affect this induction in various types of human endothelial cells. These observations may have implications for future pharmacological intervention in angiogenesis.

Recent evidence that Wnts and other genes in the Wnt signaling pathway are expressed in embryonic and adult mouse lung suggests that this pathway is important for cell fate decisions and differentiation of lung cell types. We therefore examined the expression and protein distribution of several Wnt pathway components during prenatal mouse lung development using whole-mount in situ hybridization and immunohistochemistry. Between embryonic days 10.5 and 17.5 (E10.5-E17.5), beta-catenin was localized in the cytoplasm, and often also the nucleus, of the undifferentiated primordial epithelium (PE), differentiating alveolar epithelium (AE; present from E14.5 onward), and adjacent mesenchyme. Tcf1, Lef1, Tcf3, Tcf4, sFrp1, sFrp2 and sFrp4 were also expressed in the PE, AE, and adjacent mesenchyme in specific spatio-temporal patterns.

Summary The effects of photodynamic treatment (PDT) on venules include vascular leakage accompanied by oedema formation, vasoconstriction and blood flow stasis. The goal of this study was to gain insight into the mechanism underlying these vascular events by studying one of the earliest observations after PDT, granulocyte adhesion, in an in vitro model. For this purpose human umbilical vein endothelial cells (HUVECs) preincubated with Photofrin II (PII) were illuminated with red light and incubated with neutrophils. PDT led to a dramatic change in the morphology of the endothelial cells. Clearly, neutrophils adhered to the subendothelial matrix and their adherence coincided with an increase in the percentage of exposed subendothelial matrix by the gradual contraction of endothelial cells. Furthermore, the increase in adherence was dependent on drug dose, illumination time and the time delay after PDT. The neutrophil adherence could be inhibited by anti-,B2-integrin antibodies, which suggests that the aL-, oM-or OCx-fl2 receptors of the neutrophil mediated this phenomenon. At 4°C or by preincubation of the neutrophils with staurosporin, their adherence to the subendothelial matrix exposed by PDT of endothelial cells could be prevented. Apparently, activation of the fl2-integrin receptor by interaction with the subendothelial matrix is necessary for the increased binding of neutrophils. Taken together, these in vitro findings suggest that the PDT-induced contraction of the endothelial cells permits neutrophil adherence to the subendothelial matrix. It is conceivable that a similar mechanism contributes to the initial adherence of granulocytes to the vessel wall as observed after PDT in vivo.

No abstract