Casteleyn, Christophe

Colle, Isabelle

Geerts, Anja

Heindryckx, Femke

Libbrecht, Louis

Staelens, Steven

Van Vlierberghe, Hans

Vandeghinste, Bert

Ghent University Academic Bibliography

Angiogenic chAnges in A new mouse model for hepAtocellulAr cArcinomA[1] Schoenleber SJ, et al:Prognostic role of vascular endothelial growth factor in hepatocellular carcinoma: systematic review and meta-analysis.Br J Cancer 2009, 100:1385-1392. [2] Yao DF, Wu et al:Quantitative analysis of vascular endothelial growth factor, microvascular density and their clinicopathologic features in human hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 2005, 4:220-226. [3] Hu J, et al  High expressions of vascular endothelial growth factor and platelet-derived endothelial cell growth factor predict poor prognosis in alpha-fetoprotein-negative hepatocellular carcinoma patients after curative resection. J Cancer Res Clin Oncol 2009, 135:1359-1367. [4] Poon RT, et al  High serum vascular endothelial growth factor levels predict poor prognosis after radiofrequency ablation of hepatocellular carcinoma: importance of tumor biomarker in ablative therapies. Annals of Surgical Oncology 2007, 14:1835-1845. [5] Carmeliet P et al:  Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 2001, 7:575-583.[6] Ho MC,  Placental growth factor not vascular endothelial growth factor A or C can predict the early recurrence after radical resection of hepatocellular carcinoma. Cancer Lett 2007, 250:237-249.ReFeRenCeSBACkgRounDThe increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest in this field. A vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for hepatocellular carcinoma research, (2) to assess time-dependent changes angiogenic pathways and (3) to investigate tumour growth and neo-vascularisation using state-of-the-art imaging techniques.MeTHoDS 5-week-old male mice received weekly intraperitoneal injections with n-nitrosodiethylamine (Den) (35 mg/kg bodyweight) and samples were taken at several time points. Histology, eLISA and immunohistochemical stainings were used to identify the HCC-lesions and to quantify angiogenic factors VegF and PlgF; and their receptors. HCC livers (25W) were perfused with Batson’s n°17 solution to produce vascular casts (arterial and venous). A state-of-the-art multimodal microPeT/CT was used for in vivo detection for 3D-reconstruction of the vascular casts.ReSuLTSAfter 16W of DEN-injections a mild fibrosis (F1-F2) and dysplastic lesions appear, resulting in a pre-malignant environment. An increase of angiogenic factors VegF and PlgF takes place, but not explicit enough to induce an increase in endothelial cells, which were upregulated after 20W. After 25W of Den-injections, the dysplastic lesions have progressed to vascularised exophytic tumours which are macroscopically visible and give rise to a further increase in angiogenic factors, activating the angiogenic pathway and leading to the formation of new blood vessels.  The vascular casts of HCC-livers clearly revealed the chaotic pattern and hierarchically disorganisation of tumour induced blood vessels. Arteries formed a circumferential mantle around the hepatic tumours, while the central tumour regions showed a lower arterial density. electron microscopy revealed several angiogenic spots, with mostly sprouting angiogenesis, furthermore intussusceptive  angiogenesis was also seen. gehoordConCLuSIonWhile most Den-induced models take at least one year to develop tumours, weekly injections with Den give rise to tumour occurrence after 25W. The well vascularised orthotopic tumours are a representative model for HCC and can serve as an excellent platform for the development of new therapeutic targets. The histological and serological increase of angiogenic factors (PlgF & VegF), clearly give rise to new blood vessel formation, confirmed by endothelial cell quantification and CT-reconstructions of vascular casts. High levels of VegF can predict vascular invasion of HCC and correlated with poor prognosis [1-2]. elevated VegF is also a marker for poor response to locoregional treatment and is correlated with early recurrence [3-4]. PlgF levels are known to be elevated in a variety of cancers and is associated with poor prognosis in HCC [5-6]. The up-regulation of PlgF in this Den-model supports the theory that PlgF plays an essential role in the angiogenesis of HCC.LTCSAMALHATVVCVHSVPLTCTVRKSAAMATVVCVPVHVenous cast HCC liver Arterial cast HCC liver (right lobe) Arterial cast HCC liver (left lobe)Venous cast control liverA = Aorta; L = Lungs; (L)HA = (left) hepatic artery; MA = mesenteric artery; RK = right kidney; S = spleen; SA = splenic artery; TC = truncus coeliacus; TV = tumour vessels; VC = vena cava; VP = vena porta; VH = vena hepaticaVEGFPIGFsVEGFRsVEGFRVEGFR1VEGFR2Angiogenesisup till now VegF, was one of the key targets in tumour induced angiogenesis, but recently PLgF gained interest because its association with pathological conditions. Both VegF and PLgF can bind to VegFR1, though only the latter can induce signal transduction leading to neo-vascularisation and arteriogenesis. Soluble VegF receptors are known to capture VegF without inducing signal transduction and therefore serve as decoy receptors.Soluble vascular endothelial growth factor receptor-1 (sVegFR1), a naturally occurring soluble form of the VegF receptor1, is an important negative counterpart of the VegF signalling pathway. Therefore, we measured the serum levels of sVegFR1. There was a slight increase of sVegFR1 at 30W DEN treatment, but no significant difference was seen between controls and 25W Denprotein levels of VegF in liver tissue. Asterisks (*) represent the significant P-values of the control group compared to Den-groups (* = p<0,05; ** = p<0,01 and *** = p < 0,001). These data support the theory that PLgF plays an important role in the pathogenesis of HCC.protein levels of PlgF in liver tissue, the vast up-regulation of PlgF in liver tissue after 30W Den veils the 2.4x increase at 25W Arterial mantle MicrovasculatureSprouting angiogenesis Intussusceptive angiogenesisFemke Heindryckx1, Bert Vandeghinste2, Louis Libbrecht3, Christophe Casteleyn4, Steven Staelens2,  Anja geerts1, Isabelle Colle1 & Hans Van Vlierberghe11 Department of gastroenterology and Hepatology, ghent university Hospital, ghent, Belgium2 Medical Signal and Image Processing, ghent university-IBBT, ghent, Belgium 3 Department of Pathology, ghent university Hospital, ghent, Belgium4 Department of Morphology, Faculty of Veterinary Medicine, ghent university, ghent, Belgium Femke.Heindryckx@ugent.be Angiogenic changes in a new mouse model for hepatocellular carcinomaFemke Heindryckx1, Bert Vandeghinste2, Louis Libbrecht3, Christophe Casteleyn4, Steven Staelens2,  Anja Geerts1, Isabelle Colle1 & Hans Van Vlierberghe11 Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium 2 Medical Signal and Image Processing, Ghent University-IBBT, Ghent, Belgium  3 Department of Pathology, Ghent University Hospital, Ghent, Belgium 4 Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Ghent, BelgiumFemke.Heindryckx@UGent.be [1] Schoenleber SJ, et al:Prognostic role of vascular endothelial growth factor in hepatocellular carcinoma: systematic review and meta-analysis.Br J Cancer 2009,  100:1385-1392. [2] Yao DF, Wu et al:Quantitative analysis of vascular endothelial growth factor, microvascular density and their clinicopathologic features in human hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 2005, 4:220-226. [3] Hu J, et al High expressions of vascular endothelial growth factor and platelet-derived endothelial cell growth factor predict poor prognosis in alpha-fetoprotein-negative hepatocellular carcinoma patients after curative resection. J Cancer Res Clin Oncol 2009, 135:1359-1367. [4] Poon RT, et al High serum vascular endothelial growth factor levels predict poor prognosis after radiofrequency ablation of hepatocellular carcinoma: importance of tumor biomarker in ablative therapies. Annals of Surgical Oncology 2007, 14:1835-1845. [5] Carmeliet P et al: Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 2001,  7:575-583.[6] Ho MC, Placental growth factor not vascular endothelial growth factor A or C can predict the early recurrence after radical resection of hepatocellular carcinoma. Cancer Lett 2007,  250:237-249.ReFeRenCeSBACkGRoUnDThe increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest in this field. A vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for hepatocellular carcinoma research, (2) to assess time-dependent changes angiogenic pathways and (3) to investigate tumour growth and neo-vascularisation using state-of-the-art imaging techniques.MeTHoDS 5-week-old male mice received weekly intraperitoneal injections with n-nitrosodiethylamine (Den) (35 mg/kg bodyweight) and samples were taken at several time points. Histology, eLISA and immunohistochemical stainings were used to identify the HCC-lesions and to quantify angiogenic factors VeGF and PlGF; and their receptors. HCC livers (25W) were perfused with Batson’s n°17 solution to produce vascular casts (arterial and venous).  A state-of-the-art multimodal microPeT/CT was used for in vivo detection for 3D-reconstruction of the vascular casts.ReSULTSAfter 16W of Den-injections a mild fibrosis (F1-F2) and dysplastic lesions appear, resulting in a pre-malignant environment. An increase of angiogenic factors VeGF and PlGF takes place, but not explicit enough to induce an increase in endothelial cells, which were upregulated after 20W. After 25W of Den-injections, the dysplastic lesions have progressed to vascularised exophytic tumours which are macroscopically visible and give rise to a further increase in angiogenic factors, activating the angiogenic pathway and leading to the formation of new blood vessels.  The vascular casts of HCC-livers clearly revealed the chaotic pattern and hierarchically disorganisation of tumour induced blood vessels. Arteries formed a circumferential mantle around the hepatic tumours, while the central tumour regions showed a lower arterial density. electron microscopy revealed several angiogenic spots, with mostly sprouting angiogenesis, furthermore intussusceptive  angiogenesis was also seen. gehoordVEGFPIGFsVEGFRsVEGFRVEGFR1VEGFR2AngiogenesisUp till now VeGF, was one of the key targets in tumour induced angiogenesis, but recently PLGF gained interest because its association with pathological conditions. Both VeGF and PLGF can bind to VeGFR1, though only the latter can induce signal transduction leading to neo-vascularisation and arteriogenesis. Soluble VeGF receptors are known to capture VeGF without inducing signal transduction and therefore serve as decoy receptors.ConCLUSIonWhile most Den-induced models take at least one year to develop tumours, weekly injections with Den give rise to tumour occurrence after 25W. The well vascularised orthotopic tumours are a representative model for HCC and can serve as an excellent platform for the development of new therapeutic targets. The histological and serological increase of angiogenic factors (PlGF & VeGF), clearly give rise to new blood vessel formation, confirmed by endothelial cell quantification and CT-reconstructions of vascular casts. High levels of VeGF can predict vascular invasion of HCC and correlated with poor prognosis [1-2]. elevated VeGF is also a marker for poor response to locoregional treatment and is correlated with early recurrence [3-4]. PlGF levels are known to be elevated in a variety of cancers and is associated with poor prognosis in HCC [5-6]. The up-regulation of PlGF in this Den-model supports the theory that PlGF plays an essential role in the angiogenesis of HCC.Soluble vascular endothelial growth factor receptor-1 (sVeGFR1), a naturally occurring soluble form of the VeGF receptor1, is an important negative counterpart of the VeGF signalling pathway. Therefore, we measured the serum levels of sVeGFR1. There was a slight increase of sVeGFR1 at 30W Den treatment, but no significant difference was seen between controls and 25W DenArterial mantleMicrovasculatureprotein levels of VeGF in liver tissue. Asterisks (*) represent the significant P-values of the control group compared to Den-groups (* = p<0,05; ** = p<0,01 and *** = p < 0,001). These data support the theory that PLGF plays an important role in the pathogenesis of HCC.LTCSAMALHATVVCVHSVPLTCTVRKSAAMATVVCVPVHVenous cast HCC liverArterial cast HCC liver (right lobe) Arterial cast HCC liver (left lobe)Venous cast control liverA = Aorta; L = Lungs; (L)HA = (left) hepatic artery; MA = mesenteric artery; RK = right kidney; S = spleen; SA = splenic artery; TC = truncus coeliacus; TV = tumour vessels; VC = vena cava; VP = vena porta; VH = vena hepaticaprotein levels of PlGF in liver tissue, the vast up-regulation of PlGF in liver tissue after 30W Den veils the 2.4x increase at 25W Sprouting angiogenesisIntussusceptive angiogenesis

Angiogenic changes in a new mouse model for hepatocellular carcinoma

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Angiogenic changes in a new mouse model for hepatocellular carcinoma

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Ghent University Academic Bibliography