Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo.

We have tested the effect of thrombin on endothelial cell tube formation in vitro and angiogenesis in vivo. Thrombin induces the differentiation of endothelial cells into capillary structures in a dose-dependent fashion (0.1-0.3 units thrombin/ml) on Matrigel, a laminin-rich reconstituted basement membrane matrix. At higher thrombin concentrations (1.0 unit/ml), a suppression of tube formation is evident, probably due to downregulation (desensitization) of the thrombin receptor. D-Phe-Pro-Arg-CH2Cl-thrombin is without effect when used alone, but it abolishes the tube-promoting effect of thrombin when used in combination with thrombin, indicating the involvement of the catalytic site of thrombin. Activation of protein kinase C (PKC) seems to be the transduction mechanism involved in the stimulation of tube formation by thrombin. Ro-318220 (3 micrograms/ml), a specific inhibitor of PKC, completely abolishes the stimulatory effect of thrombin. In the in vivo Matrigel system of angiogenesis, there is a 10-fold increase in endothelial cell infiltration in response to thrombin. These results provide evidence for the angiogenesis-promoting effect of thrombin in vivo and the induction by thrombin of the angiogenic phenotype of endothelial cells in vitro in the absence of other cell types such as smooth muscle cells, pericytes, and inflammatory cells.

Validation of collagenous protein synthesis as an index for angiogenesis with the use of morphological methods.

A method providing a biochemical index for the evaluation of promoters or inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) is here described and validated. This method is based on the determination of collagenous protein synthesis which takes place during new vessel formation. Validation was done by comparing collagenous protein synthesis to morphological methods of determining vascular density either by counting the number of vessels intersecting three concentric rings or by computer-assisted image analysis. Five compounds which promote or inhibit angiogenesis in the CAM were used for this purpose. The protein kinase C activator 4-beta-phorbol 12-myristate 13-acetate and alpha-thrombin increased collagenous protein synthesis and these results correlated with those obtained by using the two morphological methods mentioned above. Similarly, the inhibitors of angiogenesis, Ro318220, tricyclodecan-9-yl xanthate (D609), and 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), reduced collagenous protein synthesis and vascular density (determined by image analysis or by counting the number of vessels intersecting three concentric rings) to a comparable degree. These results indicate that collagenous protein synthesis can be used as a reliable, reproducible, and unbiased index of angiogenesis in the chick chorioallantoic membrane.

Inhibitors of basement membrane collagen synthesis prevent endothelial cell alignment in matrigel in vitro and angiogenesis in vivo.

BACKGROUND: The formation of a basement membrane is the last step in the development of a new blood vessel. Matrigel, a laminin-rich reconstituted basement membrane matrix induces the differentiation of endothelial cells into capillary-like structures. EXPERIMENTAL DESIGN: The effect of inhibitors of basement membrane collagen synthesis, tricyclodecan-9-yl xanthate (D609) and 8,9-dihydroxy-7-methyl-benzo[b] quinolizinium bromide (GPA 1734), was investigated on endothelial cell tube formation on Matrigel in vitro and in an angiogenesis assay in C57 black mice in vivo. RESULTS: D609 and GPA 1734 caused a dose-dependent decrease in tube formation in vitro with complete inhibition at 50 micrograms/ml for D609 and 15 micrograms/ml for GPA 1734. The inhibitory effect on capillary tube formation by both agents was reversible. Tube formation correlated well with collagenous protein biosynthesis. Parallel studies on endothelial cells cultured on plastic indicate that cell viability, proliferation, attachment, and morphology were not affected by the presence of these collagen inhibitors at doses that blocked tube formation and collagen biosynthesis. D609 and GPA 1734 also inhibited endothelial cell infiltration in response to SIKVAV in an in vivo angiogenesis model system. CONCLUSIONS: These results indicate that newly synthesised collagen is a prerequisite for expression of the endothelial cell phenotype for tube formation and that prevention of collagenous protein biosynthesis inhibits tube formation and angiogenesis in vivo.

Opposing effects on modulation of angiogenesis by protein kinase C and cAMP-mediated pathways.

The role of cAMP-mediated pathway in modulating angiogenesis was investigated. We have shown previously that activators of protein kinase C (PKC) caused a marked increase in angiogenesis, while the specific inhibitor of PKC, Ro 318220 suppressed angiogenesis. Here we show that forskolin, which activates adenylate cyclase and elevates the intracellular levels of cAMP, and the Sp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Sp-cAMPS), caused a dose-dependent suppression of collagenous protein biosynthesis and angiogenesis in the chick chorioallantoic membrane system (CAM). The opposite modulation of angiogenesis by activators of PKC and elevated cAMP levels was further confirmed by the suppression of 4 beta-phorbol-12-myristate-13-acetate (4 beta-PMA)-stimulated angiogenesis by either forskolin or Sp-cAMPS. On the contrary, the Rp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Rp-cAMPS), which antagonises endogenous cAMP biochemical actions, had no effect on angiogenesis alone and did not suppress 4 beta-PMA stimulated angiogenesis. However, Rp-cAMPS antagonised the effect of forskolin and Sp-cAMPS on 4 beta-PMA induced angiogenesis. Similar results were obtained in the human umbilical vein endothelial cell tube formation assay. In this system, the PKC inhibitor, Ro 318220, caused a dose-dependent inhibition of 4 beta-PMA reversed this effect. Also, forskolin and Sp-cAMPS caused an inhibition in tube formation. These results indicate that increased levels of intracellular cAMP have a negative effect in normal angiogenesis and cause a large reduction of the promotion of angiogenesis resulting from PKC activation.