OBJECTIVETo investigate the dynamic features of angiogenesis in residual tumors after high intensity focused ultrasound (HIFU),and to determine the temporal effect and mechanism of hypoxia inducible factor-2 alpha (HIF-2a) in the angiogenic process of residual tumors.

METHODSXenograft tumors of HepG2 cells were generated by subcutaneously inoculating athymic BALB/c nu/nu mice with the hepatoma cells.About 30 days after inoculation,all mice (except in the control group) were treated by HIFU and assigned randomly to the following 7 groups according to various time intervals post-treatment:1st,3rd,5th day and 1st,2nd,3rd,4th week when the residual tumor tissues were obtained from the experimental groups.Protein levels of HIF-2a and vascular growth factor A (VEGF-A) were quantified by immunohistochemistry and western blotting,and mRNA levels were measured by (real-time quantitative) qPCR. Microvascular density (MVD) was calculated by counting the CD31-positive vascular endothelial cells identified by means of an immunohistochemical staining method.

RESULTSCompared with results from the control group,the protein and mRNA levels of HIF-2a expression reached the highest level in the experimental mice at the 2nd week (P=0.000 and P < 0.01 respectively),and were decreased thereafter(3rd week and 4th week, P=0.000 and P < 0.05).VEGF-A expression in the residual tumor tissues group that received HIFU was significantly decreased,compared with the control group,at all time points uPto 1 week (all P=0.000 and P < 0.01),but the levels increased compared to controls in the 2nd through 4th week (all P=0.000, P < 0.05). Similar results were obtained for MVD.

CONCLUSIONHIFU treatment can inhibit angiogenesis in residual hepatoma tissues in the short-term (1 to 2 weeks post-treatment) in mice with hepatocellular carcinoma,but can promote angiogenesis overtime (2 to 4 weeks post-treatment); the angiogenic process may involve the HIF-2α/VEGFA pathway.

Animals ; Blotting, Western ; Carcinoma, Hepatocellular ; pathology ; Hep G2 Cells ; High-Intensity Focused Ultrasound Ablation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Immunohistochemistry ; Liver Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo study the changes in hypoxia-inducible factor (HIF1α, HIF2α) in the residual tumor cells in nude mice bearing hepatocellular carcinoma (HCC) following treatment with high-intensity focused ultrasound (HIFU).

METHODSThirty nude mice bearing human HCC received treatment with HIFU. At 1, 3, and 5 days and 1 and 2 weeks after the treatment, the mice were examined for pathological changes of the residual tumor with HE staining; SP immunohistochemistry, Western blotting and real-time quantitative PCR were used to detect the protein and mRNA expressions of HIF1α and HIF2α in the tumor.

RESULTSHE staining revealed the presence of residual tumor cells and large necrotic areas after the treatment. Immunohistochemistry showed a gradual increment of HIF1α protein and mRNA expressions after the treatment, reaching the peak level at 3 days (P<0.05) followed by progressive reduction at 5 days and 1 and 2 weeks. HIF2α expressions at either the protein or mRNA levels exhibited no significant changes within 3 days after the treatment (P>0.05) but increased significantly at 5 days and 1 and 2 weeks (P<0.05).

CONCLUSIONThe changes of HIF1α and HIF2α in the residual tumor after HIFU treatment in nude mice bearing HCC can be associated with tumor cell apoptosis and angiogenesis after the treatment.

Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; therapy ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; therapy ; Mice ; Neoplasm, Residual ; metabolism ; pathology ; Ultrasonic Therapy ; methods

To prepare RGD peptide modified perfluorohexane ( PFH ) polymer nanoparticles RGD‐PFH‐NPs and investigate its basic characteristics ,targeting ability and combine with low‐intensity focused ultrasound ( LIFU ) for ultrasonic imaging in vivo and vitro . Methods Targeted nanoparticles RGD‐PFH‐NPs were prepared by double emulsifying method and carbodiimide method . T heir morphology and distribution were observed . T he particle size ,zeta potential and connection probability were measured . T he phase‐changed properties and the LIFU‐induced imaging ability in contrast‐enhanced ultrasound mode of RGD‐PFH‐NPs were investigated in vivo and vitro . T he tagetability of nanoparticles to human gastric cancer cell line M GC803 and tumor‐bearing nude mouse were observed through targeting group and non‐targeting group . Results T he prepared sample was milky w hite suspension liquid . T he RGD‐PFH‐NPs were spherical uniform size ,good dispersion w hen observed through the optical microscope and transmission electron microscopy . T he particle size was ( 259 .3 ± 42 .6) nm and the Zeta potential was ( －17 .6+5 .4) mV . T he connection probability of RGD peptide was 89 .13% . With 70℃ water bath and LIFU stimulation RGD‐PFH‐NPs can remarkably change phase and show good imaging performance in both conventional ultrasound and contrast‐enhanced ultrasound mode in vivo and vitro . The connection probability to M GC803 cells in targeting group ( RGD‐PFH‐NPs ) and the non‐targeting group( PFH‐NPs) were 82 .59% and 2 .96% . T he accumulation of nanoparticles in the RGD‐PFH‐NPs group in tumor tissues was significantly higher than that in the non‐targeted PFH‐NPs group( P) . Conclusions The constructed nanoparticles RGD‐PFH‐NPs ,providing contrast‐enhanced ultrasonic imaging and excellent targeting ability to human gastric cancer cells M GC803 and gastric cancer tissue ,is expected to become a new type of gastric cancer targeted ultrasound contrast agent .

Objective To prepare RGD peptide modified perfluorohexane ( PFH ) polymer nanoparticles RGD-PFH-NPs and investigate its basic characteristics ,targeting ability and combine with low-intensity focused ultrasound ( LIFU ) for ultrasonic imaging in vivo and vitro . Methods Targeted nanoparticles RGD-PFH-NPs were prepared by double emulsifying method and carbodiimide method . Their morphology and distribution were observed . The particle size ,zeta potential and connection probability were measured . The phase-changed properties and the LIFU-induced imaging ability in contrast-enhanced ultrasound mode of RGD-PFH-NPs were investigated in vivo and vitro . The tagetability of nanoparticles to human gastric cancer cell line MGC803 and tumor-bearing nude mouse were observed through targeting group and non-targeting group . Results The prepared sample was milky white suspension liquid . The RGD-PFH-NPs were spherical uniform size ,good dispersion when observed through the optical microscope and transmission electron microscopy . The particle size was ( 259 .3 ± 42 .6) nm and the Zeta potential was ( -17 .6+5 .4) mV . The connection probability of RGD peptide was 89 .13％ . With 70℃ water bath and LIFU stimulation RGD-PFH-NPs can remarkably change phase and show good imaging performance in both conventional ultrasound and contrast-enhanced ultrasound mode in vivo and vitro . The connection probability to MGC803 cells in targeting group ( RGD-PFH-NPs ) and the non-targeting group( PFH-NPs)were 82 .59％ and 2 .96％ . The accumulation of nanoparticles in the RGD-PFH-NPs group in tumor tissues was significantly higher than that in the non-targeted PFH-NPs group( P) . Conclusions The constructed nanoparticles RGD-PFH-NPs ,providing contrast-enhanced ultrasonic imaging and excellent targeting ability to human gastric cancer cells MGC803 and gastric cancer tissue ,is expected to become a new type of gastric cancer targeted ultrasound contrast agent .

Objective To study the changes in hypoxia-inducible factor (HIF1α, HIF2α) in the residual tumor cells in nude mice bearing hepatocellular carcinoma (HCC) following treatment with high-intensity focused ultrasound (HIFU). Methods Thirty nude mice bearing human HCC received treatment with HIFU. At 1, 3, and 5 days and 1 and 2 weeks after the treatment, the mice were examined for pathological changes of the residual tumor with HE staining; SP immunohistochemistry, Western blotting and real-time quantitative PCR were used to detect the protein and mRNA expressions of HIF1α and HIF2α in the tumor. Results HE staining revealed the presence of residual tumor cells and large necrotic areas after the treatment. Immunohistochemistry showed a gradual increment of HIF1αprotein and mRNA expressions after the treatment, reaching the peak level at 3 days (P<0.05) followed by progressive reduction at 5 days and 1 and 2 weeks. HIF2αexpressions at either the protein or mRNA levels exhibited no significant changes within 3 days after the treatment (P>0.05) but increased significantly at 5 days and 1 and 2 weeks (P<0.05). Conclusion The changes of HIF1αand HIF2αin the residual tumor after HIFU treatment in nude mice bearing HCC can be associated with tumor cell apoptosis and angiogenesis after the treatment.

Objective To study the changes in hypoxia-inducible factor (HIF1α, HIF2α) in the residual tumor cells in nude mice bearing hepatocellular carcinoma (HCC) following treatment with high-intensity focused ultrasound (HIFU). Methods Thirty nude mice bearing human HCC received treatment with HIFU. At 1, 3, and 5 days and 1 and 2 weeks after the treatment, the mice were examined for pathological changes of the residual tumor with HE staining; SP immunohistochemistry, Western blotting and real-time quantitative PCR were used to detect the protein and mRNA expressions of HIF1α and HIF2α in the tumor. Results HE staining revealed the presence of residual tumor cells and large necrotic areas after the treatment. Immunohistochemistry showed a gradual increment of HIF1αprotein and mRNA expressions after the treatment, reaching the peak level at 3 days (P<0.05) followed by progressive reduction at 5 days and 1 and 2 weeks. HIF2αexpressions at either the protein or mRNA levels exhibited no significant changes within 3 days after the treatment (P>0.05) but increased significantly at 5 days and 1 and 2 weeks (P<0.05). Conclusion The changes of HIF1αand HIF2αin the residual tumor after HIFU treatment in nude mice bearing HCC can be associated with tumor cell apoptosis and angiogenesis after the treatment.