Neutrophil-Mediated Proteolysis of Thrombospondin-1 Promotes Platelet Adhesion and String Formation.

Thrombospondin-1 (TSP-1) is primarily expressed by platelets and endothelial cells (ECs) and rapidly released upon their activation. It functions in haemostasis as a bridging molecule in platelet aggregation, by promoting platelet adhesion to collagen and by protecting von Willebrand factor strings from degradation. In blood of patients undergoing surgery and in co-cultures of neutrophils with platelets or ECs, we observed proteolysis of the 185 kDa full-length TSP-1 to a 160-kDa isoform. We hypothesized that TSP-1 processing may alter its haemostatic properties. Selective enzyme inhibitors in co-cultures revealed that neutrophil proteases elastase and cathepsin G mediate TSP-1 processing. The cut site of cathepsin G was mapped to TSP-1 amino acids R237/T238 by Edman sequencing. Formation of neutrophil extracellular traps protected TSP-1 from complete degradation and promoted controlled processing to the 160-kDa isoform. Haemostatic properties were tested by platelet aggregation, adhesion, coagulation and string formation under flow. Platelets from TSP-1 deficient mice did not differ from wild-type in platelet aggregation but showed severe impairment of platelet adhesion to collagen and string formation under flow. Reconstitution experiments revealed that the 160-kDa TSP-1 isoform was markedly more potent than the 185-kDa full-length molecule in restoring function. Thus, TSP-1 processing by neutrophil proteases yields a 160-kDa isoform which shows enhanced potency to promote platelet adhesion and string formation. This finding reveals a novel mechanism of neutrophil-mediated thrombus formation and provides first evidence for the impact of TSP-1 proteolysis on its haemostatic properties.

The VEGF rise in blood of bevacizumab patients is not based on tumor escape but a host-blockade of VEGF clearance.

Vascular endothelial growth factor (VEGF) has become a major target in cancer treatment as it promotes tumor angiogenesis. Therapy with anti-VEGF antibody bevacizumab reportedly induces high levels of circulating VEGF which may potentially contribute to resistance. Based on animal or computational models, mechanisms of VEGF induction by bevacizumab have been proposed but not verified in the clinical setting. Hence, we evaluated sixty patients with colorectal cancer metastases for changes in plasma VEGF during neoadjuvant/conversion and adjuvant chemotherapy with or without bevacizumab. VEGF expression was assessed in tissue sections of liver metastases. The VEGF source was investigated with in vitro cultures of tumor, endothelial cells, fibroblasts and platelets, and potential protein stabilization due to anti-VEGF therapy was addressed. A VEGF rise was observed in blood of bevacizumab patients but not in chemotherapy controls, and VEGF was found to be largely complexed by the antibody. A comparable VEGF increase occurred in the presence (neoadjuvant) and absence of the tumor (adjuvant). Accordingly, VEGF expression in tumor tissue was not determined by bevacizumab treatment. Investigations with isolated cell types did not reveal VEGF production in response to bevacizumab. However, antibody addition to endothelial cultures led to a dose-dependent blockade of VEGF internalization and hence stabilized VEGF in the supernatant. In conclusion, the VEGF rise in cancer patients treated with bevacizumab is not originating from the tumor. The accumulation of primarily host-derived VEGF in circulation can be explained by antibody interference with receptor-mediated endocytosis and protein degradation. Thus, the VEGF increase in response to bevacizumab therapy should not be regarded as a tumor escape mechanism.

Chemotherapy of colorectal liver metastases induces a rapid rise in intermediate blood monocytes which predicts treatment response.

We have previously reported that intermediate monocytes (CD14(++)/CD16(+)) were increased in colorectal cancer (CRC) patients, while the subset of pro-angiogenic TIE2-expressing monocytes (TEMs) was not significantly elevated. This study was designed to evaluate changes in frequency and function of intermediate monocytes and TEMs during chemotherapy and anti-angiogenic cancer treatment and their relation to treatment response. Monocyte populations were determined by flow cytometry in 60 metastasized CRC (mCRC) patients who received neoadjuvant chemotherapy with or without bevacizumab. Blood samples were taken before treatment, after two therapy cycles, at the end of neoadjuvant therapy and immediately before surgical resection of liver metastases. Neoadjuvant treatment resulted in a significant increase in circulating intermediate monocytes which was most pronounced after two cycles and positively predicted tumor response (AUC = 0.875, p = 0.005). With a cut-off value set to 1% intermediate monocytes of leukocytes, this parameter showed a predictive sensitivity and specificity of 75% and 88%. Anti-angiogenic therapy with bevacizumab had no impact on monocyte populations including TEMs. In 15 patients and six healthy controls, the gene expression profile and the migratory behavior of monocyte subsets was evaluated. The profile of intermediate monocytes suggested functions in antigen presentation, inflammatory cytokine production, chemotaxis and was remarkably stable during chemotherapy. Intermediate monocytes showed a preferential migratory response to tumor-derived signals in vitro and correlated with the level of CD14(+)/CD16(+) monocytic infiltrates in the resected tumor tissue. In conclusion, the rapid rise of intermediate monocytes during chemotherapy may offer a simple marker for response prediction and a timely change in regimen.

Monocytes with angiogenic potential are selectively induced by liver resection and accumulate near the site of liver regeneration.

BACKGROUND: Monocytes reportedly contribute to liver regeneration. Three subsets have been identified to date: classical, intermediate, non-classical monocytes. The intermediate population and a subtype expressing TIE2 (TEMs) were suggested to promote angiogenesis. In a clinical setting, we investigated which monocyte subsets are regulated after liver resection and correlate with postoperative liver function. METHODS: In 38 patients monocyte subsets were evaluated in blood and subhepatic wound fluid by flow cytometry before and 1-3 days after resection of colorectal liver metastases. The monocyte-regulating cytokines macrophage colony stimulating factor (M-CSF), transforming growth factor beta 1 (TGFß1), and angiopoietin 2 (ANG-2) were measured in patient plasma by ELISA. C-reactive protein (CRP) and liver function parameters were retrieved from routine hospital analyses. RESULTS: On post-operative day (POD) 1 blood monocytes shifted to significantly elevated levels of intermediate monocytes. In wound fluid, a delayed surge in intermediate monocytes was detected by POD 3. Furthermore, TEMs were highly enriched in wound fluid as compared to circulation. CRP and M-CSF levels were substantially increased in patient blood after surgery and correlated significantly with the frequency of intermediate monocytes. In addition, liver function parameters showed a significant association with intermediate monocyte levels on POD 3. CONCLUSIONS: The reportedly pro-angiogenic subsets of monocytes are selectively increased upon liver resection and accumulate next to the site of liver regeneration. As previously proposed by in vitro experiments, the release of CRP and M-CSF may trigger the induction of intermediate monocytes. The correlation with liver parameters points to a functional involvement of these monocyte populations in liver regeneration which warrants further investigation.

Thrombocytes Correlate with Lymphangiogenesis in Human Esophageal Cancer and Mediate Growth of Lymphatic Endothelial Cells In Vitro.

Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC) were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC), and 136 squamous cell carcinomas (SCC). Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs) were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC) were evident in 82 samples (25.6%), and vascular thrombocytic clusters (VTC) in 56 (17.5%). STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001), PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test), and VTC with shorter DFS in in SCC (p = 0.025, Breslow test). In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.

Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.

BACKGROUND: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. METHODS: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. RESULTS: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF-4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P=0.006; P< 0.001). CONCLUSION: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.