Continuous thrombin infusion leads to a bleeding phenotype in sheep.

BACKGROUND: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo. METHODS: Thrombin (0.0004-0.42 IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42 IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments). RESULTS: Thrombin at an infusion rate of 0.42 IU/kg/min decreased fibrinogen levels by 75% (p<0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p<0.05) and decreased the activity of the coagulation factors VIII and XIII (p<0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p<0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism. CONCLUSION: Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.

An experimental model to study isolated effects of thrombin in vivo.

BACKGROUND: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo. METHODS: Thrombin (0.05-0.9U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n=38). The study consisted of three parts: dose escalation (n=21), dose verification (n=5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n=12). RESULTS: A thrombin dose of 0.9U/kg/min decreased platelet counts by 70% compared to the control group (median 230×10^9/L vs. 752×10^9/L; p=0.041). In accordance, infusion of 0.9U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56mg/dl vs. 220mg/dl; p=0.046). Cumulative thrombin doses of ≥0.1U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥0.15U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9U/kg/min was associated with a 3 to 6.5 -fold increase in IL-6 levels (139-306pg/ml vs. 47pg/ml, p<0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNF-alpha). In addition, lepirudin prevented thrombin- induced thrombocytopenia. CONCLUSION: Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines.

VEGF-C expressing tumor-associated macrophages in lymph node positive breast cancer: impact on lymphangiogenesis and survival.

BACKGROUND: The ability of malignant tumors to metastasize presents a severe challenge in cancer treatment. Lymphatic vessels provide one of the main routes for tumor-metastasis on the way to regional lymph nodes. Increasing evidence suggests that inflammatory cells play an important role in tumor-associated angiogenesis and lymphangiogenesis. Recent data show that a specialized sub fraction of tumor-associated macrophages (TAMs) expressing the lymphoangiogenic growth factors vascular endothelial growth factor-C and -D (VEGF-C/D) at the tumor site, is related to lymphangiogenesis, lymphovascular invasion, and lymph node metastasis. Aim of this study was to clear the role of VEGF-C/D expressing TAMs in invasive breast cancer. METHODS: One hundred-seven cases of lymph node positive invasive breast cancer were included into the study. Lymphatic microvessel density (LMVD), lymphovascular invasion (LVI), peritumoral inflammatory reaction (PI), and VEGF-C expression in tumors (VEGF-C(T)) and TAMs (VEGF-C(C)) were evaluated by immunohistochemistry and in situ hybridization. RESULTS: Significant associations were seen between LMVD and LVI, LMVD and VEGF-C(T), and between VEGF-C(T) and VEGF-C(C). Further significant correlations were evaluated between VEGF-C(C)/VEGF-C(T) and PI as well as between PI and LVI. LVI remained an independent prognostic factor for disease-free survival and overall survival. CONCLUSIONS: Our data provide evidence that the peritumoral inflammatory reaction and VEGF-C expressing TAMs may play an important role in tumor lymphangiogenesis and lymphovascular invasion in invasive breast cancer, implying new potential anti-tumor targets.

Prognostic value of lymphangiogenesis and lymphovascular invasion in invasive breast cancer.

OBJECTIVE: The aim of this study was to investigate the prognostic relevance of lymphangiogenesis and lymphovascular invasion in a large cohort of breast cancer patients. INTRODUCTION: Invasion of tumor cells into blood and lymphatic vessels is one of the critical steps for metastasis. The presence or absence of lymph node metastasis is one of the main decision criteria for further therapy. One shortcoming of previous morphologic studies was the lack of specific markers that could exact discriminate between blood and lymphatic vessels. The aim of this study was to evaluate the prognostic relevance of lymphangiogenesis and lymphovascular invasion in breast cancer patients. METHODS: We investigated 374 tissue specimens of patients suffering from invasive breast cancer by immunostaining for the lymphatic endothelial specific marker podoplanin. Lymphangiogenesis, quantified by evaluating the lymphatic microvessels density (LMVD), and lymphovascular invasion (LVI) were correlated with various clinical parameters and prognostic relevance. RESULTS: LMVD correlated significantly with LVI (P = 0.001). LVI was associated significantly with a higher risk for developing lymph-node metastasis (P = 0.004). Calculating the prognostic relevance, LVI presented as an independent prognostic parameter for disease free as well as overall survival (P = 0.001, and P = 0.001, respectively). CONCLUSION: Our data provide evidence that the biologic system of lymphangiogenesis constitutes a potential new target for development of anti-breast cancer therapeutic concepts. Our results further suggest that young, premenopausal patients with low differentiated breast tumors and high LMVD and LVI would, in particular, benefit from lymphangiogenesis-associated therapeutic strategies.

Overexpression of Id-1 is associated with poor clinical outcome in node negative breast cancer.

Id-1 is an important regulator of cellular growth and differentiation and controls malignant progression of breast cancer cells. The aim of our study was to assess the clinical impact of Id-1 expression in breast cancer, i.e., its potential impact on prognosis and prediction of treatment response. Id-1 protein expression was determined immunohistochemically in 191 patients with lymph-node negative breast cancer, and univariate and multivariate survival analysis was carried out. Fifteen (7.9%) specimens showed strong expression, 75 (39.3%) moderate, 55 (28.8%) weak expression and 46 (24.1%) cases no expression of Id-1. Patients with strong or moderate Id-1 expression had a significant shorter overall (p = 0.003, Cox regression) and disease-free survival (p = 0.01, Cox regression) compared to those with absent or low expression. Progesterone receptor density was significantly higher in breast cancers with absent/low Id-1 expression compared to those with moderate/strong expression (p < 0.001, t-test). Id-1 expression was significantly stronger in cases positive for p16(INK4a) expression compared to those negative for p16 (p = 0.049, Mann-Whitney test). The influence of Id-1 on clinical outcome seems much stronger in patients with negative estrogen receptor status compared to those with positive status, who received receptor antagonists as adjuvant therapy in most cases. Overexpression of Id-1 protein represents a strong independent prognostic marker in node negative breast cancer, and future therapies inhibiting Id-1 expression might be beneficial for these patients. Our results also suggest that due to the apparent interaction of Id-1 with the steroid-receptor system in breast cancer, hormonal therapies might influence Id-1 expression and its impact on clinical outcome.