Nikos E. Tsopanoglou

On the Mechanism of Thrombin-induced Angiogenesis POTENTIATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR ACTIVITY ON ENDOTHELIAL CELLS BY UP-REGULATION OF ITS RECEPTORS

Many of the cellular actions of thrombin may contribute to the angiogenesis-promoting effect of thrombin reported previously. In this study, we investigated the interaction between thrombin and vascular endothelial growth factor (VEGF), the specific endothelial cell mitogen and key angiogenic factor. Exposure of human umbilical vein endothelial cells to thrombin sensitizes these cells to the mitogenic activity of VEGF. This thrombin-mediated effect is specific, dose-dependent and requires the activated thrombin receptor. Quantitative reverse transcription- polymerase chain reaction analysis reveals a time- and dose-dependent up-regulation of mRNA for VEGF receptors (KDR and flt-1). Optimal thrombin concentration for maximal expression of mRNA for KDR is 1.5 IU/ml (170% over controls) and appears 8–12 h after thrombin stimulation. Nuclear run-on experiments demonstrate that the up-regulation of KDR mRNA by thrombin occurred at the transcriptional level. In addition, functional protein of KDR receptor is increased to about 200% over control after 12 h of thrombin treatment. The up-regulation of KDR and flt-1 mRNA is also mimicked by the thrombin receptor activating peptide. These findings could explain at least in part the potent angiogenic action of thrombin.

Effects of thrombin/thrombosis in angiogenesis and tumour progression

Laboratory, histopathological, pharmacological and clinical evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. On the other hand, epidemiological studies provide evidence of an increased risk of cancer diagnosis following primary thromboembolism. Moreover, the metastatic ability of human breast cancer cells is correlated with the number of thrombin receptors of these cells, and thrombin treatment of B16 melanoma cells dramatically increases the number of lung metastases in rats. We have proposed that these tumour-promoting effects of thrombin can be explained by the ability of thrombin to activate angiogenesis, an essential requirement for tumour progression. Many of the cellular events involved in the angiogenic cascade can be activated by thrombin. At the molecular level, brief exposure of endothelial cells to thrombin causes an upregulation of the receptors (KDR and Flt-1) of VEGF, the key angiogenic mediator. This results in a synergistic effect of thrombin and VEGF in the activation of angiogenesis. In addition, thrombin activates cancer cells to secrete VEGF, thus causing a mutual stimulation between EC and CA cells. Cancer cells exposed to thrombin secrete metalloproteinase 92 KD and overexpress the integrin a(v)b(3), all of which are involved in tumour metastasis.

Protein kinase C involvement in the regulation of angiogenesis.

Using the chick chorioallantoic membrane as a model system for the study of angiogenesis, we have shown that promoters of protein kinase C (PKC) such as 4-beta-phorbol-12-myristate-13-acetate (4-beta-PMA) and 1,2-dioctanoyl-sn-glycerol (DiC8) stimulated angiogenesis. This effect was specific since 4-alpha-PMA and 1,2-dioleoyl-sn-glycerol, which either do not activate or cannot reach PKC, were devoid of angiogenic activity. Furthermore, Ro 31-8220, a specific inhibitor of PKC, suppressed both basal and 4-beta-PMA- or DiC8-induced angiogenesis. Similar results were obtained with the commonly used inhibitor of PKC, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine and with tricyclodecan-9-yl-xanthogenate, an antitumor agent which has been suggested to be an inhibitor of PKC. Activation of PKC may be, therefore, an important signalling pathway in the initiation and control of the angiogenic response.

Blockade of Angiogenesis by Small Molecule Antagonists to Protease-Activated Receptor-1: Association with Endothelial Cell Growth Suppression and Induction of Apoptosis

Many studies support the notion that protease-activated receptor (PAR)-1 plays a pivotal role in angiogenesis. However, direct evidence and understanding the molecular mechanisms involved were limited because PAR-1-specific antagonists have been developed only recently. In the present study, we evaluated the effects of two well characterized PAR-1 antagonists, SCH79797 ((N-3-cyclopropyl-7-{[4-(1-methylethyl)phenyl]-methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine)) and RWJ56110 [(αS)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]-α-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide], in the angiogenic cascade. These antagonists suppressed both the basic angiogenesis and that stimulated by thrombin in the chick chorioallantoic membrane model in vivo. PAR-1 antagonists also abrogated tube formation in the in vitro Matrigel system. These inhibitory effects were dose-dependent and well correlated with the inhibitory effects of SCH79797 and RWJ56110 on primary endothelial cell proliferation and on the initiation of apoptosis. PAR-1 blockage resulted in inhibition of endothelial cell growth by increasing the sub-G0/G1 fraction and reducing the percentage of cells in the S phase. Consistent with this, PAR-1 antagonists reduced incorporation of [3H]thymidine in endothelial cells and blocked the phosphorylation of extracellular signal-regulated kinases in a fashion depending specifically on PAR-1 activation. Analysis by annexin V/propidium iodide staining and poly(ADP-ribose) polymerase cleavage revealed that PAR-1 blockage increased apoptotic cell death by a mechanism involving caspases. These results provide further evidence that PAR-1 is a key receptor that mediates angiogenesis and suggest PAR-1 as target for developing antiangiogenic agents with potential therapeutic application in cancer and other angiogenesis-related diseases.

Thrombin Functions through Its RGD Sequence in a Non-canonical Conformation

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with αvβ3 and α5β1 integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187–189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Å resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Å away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.

On the mode of action of thrombin-induced angiogenesis: thrombin peptide,TP508, mediates effects in endothelial cells via αvβ3 integrin

In a previous report we have presented evidence that thrombin interacts with αvβ3 integrin in endothelial cells at the molecular and cellular level.This interaction was shown to be of functional significance in vitro and in vivo and contributed to activation of angiogenesis by thrombin. In the present study, we have used a synthetic thrombin peptide, TP508, which represents residues 183 to 200 of human thrombin.This peptide lacks the catalytic site of thrombin but contains the thrombin RGD sequence. Immobilized (surface-coated) TP508 peptide, like thrombin, supported αvβ3 integrin-dependent endothelial cell attachment and haptotactic migration. These effects were specific (a scrambled TP508 peptide was without effect), and dosedependent. The RGD sequence was essential since a modified TP508 peptide, which contained RAD sequence instead of RGD, was inactive. Immobilized TP508 peptide stimulated phos phorylation of mitogen-activated protein kinases and focal adhesion kinase, the signal transduction pathways characteristic for integrin activation. On the other hand,TP508 peptide, when in solution, did not mimic other thrombin-promoted angiogenic effects, such as that of activation gelatinase A, upregulation of expression of vascular endothelial growth factor receptor mRNA or prostacyclin PGI2 release in endothelial cells.On the contrary, soluble TP508 acted as an antagonist for the aforementioned effects of thrombin. TP508 peptide inhibited these thrombin-induced effects through a RGD and αvβ3-related mechanism.The antagonism with thrombin or thrombin receptor activating peptide was specific and involved at least in part mitogen-activated protein kinases activation. These results point to the importance of RGD sequence of thrombin in mediating effects on endothelial cells and angiogenesis.

Parstatin, the Cleaved Peptide on Proteinase-Activated Receptor 1 Activation, Is a Potent Inhibitor of Angiogenesis

The proteolytic activation by thrombin of the proteinase-activated receptor 1 unveils the tethered peptide ligand and cleaves a 41-amino acid peptide. In this report, we show that this peptide, which we have designated as “parstatin,” is a potent inhibitor of angiogenesis. Synthesized parstatin suppressed both the basic angiogenesis and that stimulated by basic fibroblast growth factor and vascular endothelial growth factor in the chick embryo model in vivo and in the rat aortic ring assay. Parstatin also abrogated endothelial cell migration and capillary-like network formation on the Matrigel and fibrin angiogenesis models in vitro. Treatment of endothelial cells with parstatin resulted in inhibition of cell growth by inhibiting the phosphorylation of extracellular signal-regulated kinases in a specific and reversible fashion and by promoting cell cycle arrest and apoptosis through a mechanism involving activation of caspases. We have shown that parstatin acts as a cell-penetrating peptide, exerting its biological effects intracellularly. The uptake into cells and the inhibitory activity were dependent on parstatin hydrophobic region. These results support the notion that parstatin may represent an important negative regulator of angiogenesis with possible therapeutic applications.

Thrombin’s central role in angiogenesis and pathophysiological processes

A plethora of endogenous modulators of angiogenesis have been identified and their roles in the molecular and cellular events that mediate and regulate angiogenesis have been proposed. In this review, we summarize the recent findings on the role of thrombin/thrombosis on angiogenesis and other related pathophysiological processes. The mechanisms by which thrombin itself and its receptor PAR1 orchestrate many cellular events through interaction with a variety of other factors and cell types are discussed. These new data point to the complexity of the regulatory processes involved in the angiogenic cascade, which may be tissue specific, and dependent upon the pathology involved. The understanding of these events may provide targets for therapeutic intervention in disease states where angiogenesis is disturbed.

Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms

Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by indirectly regulating and organizing a network of angiogenic molecules. In addition, it has been proposed that thrombin can directly activate endothelial cell proliferation. However, in this report it was shown that thrombin is a poor growth factor for human endothelial cells, and its modest mitogenic activity is mediated indirectly by the release of heparin-binding epidermal growth factor, subsequent to proteinase-activated receptor 1 (PAR1) activation. On the other hand, it was demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, pointing to a novel role of thrombin in vascular protection. Analysis by annexin V-propidium iodide double staining revealed that thrombin, specifically, promoted survival of serum-starved endothelial cells in a concentration-dependent manner. In contrast to its mitogenic effect, the anti-apoptotic effect of thrombin was largely independent of its catalytic activity and was mediated through interaction with alphanubeta3 and alpha5beta1 integrins, whereas the involvement of PAR1 was limited. These results provide new insights in understanding the role of thrombin in endothelial cell signaling and vascular biology.

On the Mechanism(s) of Thrombin Induced Angiogenesis

Promotion of tumour progression by thrombin is suggested by several clinical and laboratory observations. A plausible explanation for this effect of thrombin may be related to our previous findings that thrombin is a potent promoter of angiogenesis in the chick chorioallantoic membrane system (CAM) and in the Matrigel system in vivo. In this report we summarise the cellular and molecular actions of thrombin that could be contributing to the activation of angiogenic cascade. Treatment of endothelial cells with thrombin leads to activation of gelatinase A, which may allow for local dissolution of basement membrane, an essential first step of angiogenesis. Similarly thrombin-treated endothelial cells have diminished ability to adhere to collagen type IV and laminin. This new phenotype of endothelial cells can migrate and survive without attachment to extracellular matrix. Thrombin-treatment of endothelial cells increases the vectorial secretion of extracellular matrix proteins, a process essential at the final steps of angiogenesis. In addition, thrombin potentiates the VEGF-induced mitogenesis of endothelial cells. This can be explained by the upregulation of the VEGF receptors (KDR & flt-1) by thrombin treatment. All the aforementioned effects of thrombin are receptor mediated, dose-dependent and require only brief exposure of endothelial cells to thrombin for these actions of thrombin. The transduction mechanisms involved are via protein kinase C (PKC) and MAP-kinase pathways.