Vascular Resection During Hepatectomy for Liver Malignancies. Results from a Tertiary Center using Autologous Peritoneal Patch for Venous Reconstruction.

BACKGROUND: To evaluate early outcomes of venous reconstruction with peritoneal patch (PP) during resection for hepatic malignancies. METHODS: Since May 2015, PP was considered as the first option for venous reconstruction in the case of lateral resection. Between May 2015 and June 2019, 579 consecutive hepatectomies for malignancies were performed at our institution. Among 27 patients requiring venous resection, PP was used in 22, who were included in the present study. Data from a prospectively collected database were analysed. RESULTS: Tumour types were ten colorectal metastases (CRLM), six intrahepatic cholangiocarcinomas, four hilar cholangiocarcinomas, one hepatocellular carcinoma and one gallbladder carcinoma. Hepatectomies were major in 50% of cases. Eleven patients had hepatic vein resections, eight portal vein and three inferior vena cava. Venous reconstruction enabled resection in 12 (54.5%) patients, otherwise non-resectable. Among CRLM, the venous reconstruction allowed avoidance of major resection in eight (80%) cases. Median operative time was 456 min (range 270-960). Blood loss was a median 300 cc (range 40-1500), and blood transfusions were required in three patients (13.6%). At pathological examination, venous infiltration was confirmed in 14 (63.6%) patients. No vascular complications related to the patch were recorded. Post-operative major (Dindo III/IV) complications were observed in two (9%) patients. One patient died because of liver failure without vascular thrombosis and one due to biliary fistula complicated by arterial bleeding. Overall, post-operative mortality was 9% (2/22). CONCLUSIONS: Venous reconstruction with peritoneal patch during hepatectomy for malignancies can feasibly allow resection in otherwise unresectable patients and decrease the rate of major resection in colorectal liver metastases.

Transabdominal ultrasound evaluation of vascularity of gallbladder lesions: particularly those with wall thickening.

Gallbladder wall thickening is relatively common in clinical settings, and for appropriate diagnosis, the size, shape, internal structure, surface contour, and vascularity of the gallbladder wall must be evaluated. Morphological evaluation is the most important; however, some gallbladder lesions resemble gallbladder cancer in imaging studies, making differential diagnosis challenging. Vascular evaluation is indispensable for a precise diagnosis in these cases. In this review, we present the current status of vascular evaluation using US and diagnosis using vascular imaging for gallbladder lesions, including those presenting with wall thickening. To date, several ultrasound imaging techniques have been developed to assess vascularity, including Doppler imaging with high sensitivity, use of contrast agents, and microvascular imaging using a novel filter for Doppler imaging. Although conventional color Doppler imaging is rarely used for the diagnosis of gallbladder lesions, the efficacy of contrast-enhanced ultrasound in assessing the vascularity, enhancement pattern, or timing of enhancement/washout has been reported. Presence of multiple irregular microvessels has been speculated to indicate malignancy. However, few reports on microvessels have been published, and further studies are required for the precise diagnosis of gallbladder lesions with microvascular evaluation.

Utility of contrast-enhanced harmonic EUS in the diagnosis of malignant gallbladder polyps (with videos).

BACKGROUND: The differential diagnosis between benign and malignant polyps of the gallbladder (GB) is often challenging. OBJECTIVES: To evaluate whether contrast-enhanced harmonic EUS (CEH-EUS) might be an accurate method for discriminating malignant GB polyps from benign polyps. DESIGN: Observational study. SETTING: Tertiary care medical center. PATIENTS: Ninety-three patients with GB polyps larger than 10 mm in diameter that were detected by conventional EUS underwent CEH-EUS for evaluation of microvasculature. INTERVENTION: CEH-EUS was performed using a radial echoendoscope and the extended pure harmonic detection mode. MAIN OUTCOME MEASUREMENTS: The abilities of conventional EUS and CEH-EUS to diagnose malignant polyp were compared. Two blinded reviewers classified the perfusion images into 3 categories: diffuse enhancement, perfusion defect, or nonenhancement. The vessel images were categorized as having a regular spotty vessel, an irregular vessel, or no vessels. RESULTS: An irregular vessel pattern determined by CEH-EUS aided in the diagnosis of malignant polyps with a sensitivity and specificity of 90.3% and 96.6%, respectively. The presence of perfusion defects, determined by CEH-EUS, was calculated to diagnose malignant polyps with a sensitivity and specificity of 90.3% and 94.9%, respectively. Based on the definitely determined diagnosis, sensitivity and specificity for CEH-EUS were 93.5% and 93.2% versus 90.0% and 91.1% for conventional EUS. In 8 cases, management changed after CEH-EUS. LIMITATIONS: A tertiary medical center with a limited number of patients. CONCLUSIONS: The presence of irregular intratumoral vessels or perfusion defects seen on CEH-EUS may be sensitive and accurate predictors of malignant GB polyps. CEH-EUS offers slightly improved diagnostic accuracy compared with EUS.

Identification of CD146 expression, angiogenesis, and lymphangiogenesis as progression, metastasis, and poor-prognosis related markers for gallbladder adenocarcinoma.

Gallbladder cancers (GBC) are associated with high disease-specific mortality rates because of no means of early detection and effective therapies. In this study, we investigated CD146 expression, microvessel densities, and lymph vessel densities in 108 adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. We demonstrated that positive CD146 expression, and average microvessel and lymph vessel counts in gallbladder adenocarcinomas were significantly higher than those in peritumoral tissues, polyps, and chronic cholecystitis (ps < 0.01). Positive CD146 expression, and average microvessel and lymph vessel counts were also significantly lower in cases with well-differentiated adenocarcinoma, maximal tumor diameter <2 cm, no metastasis of lymph node, and no invasion of regional tissues than in cases with poorly differentiated adenocarcinoma, maximal tumor diameter ≥ 2 cm, metastasis in lymph nodes, and invasion of regional tissues (p < 0.05 or p < 0.01). Univariate Kaplan-Meier analysis showed that increased expression of CD146 (p = 0.056), higher average microvessel counts (p < 0.05), and lymph vessel counts (p < 0.05) were associated with decreased overall survival. Multivariate Cox regression analysis showed that average microvessel and lymph vessel counts (ps < 0.05) were independent prognostic predictors in gallbladder adenocarcinoma. Our study suggested that the elevated expression of CD146, angiogenesis, and lymphangiogenesis might be closely related to progression, invasion, metastasis, and prognosis of gallbladder adenocarcinoma.

Portal vein embolization before extended hepatectomy for biliary cancer: current technique and review of 494 consecutive embolizations.

BACKGROUNDS: Portal vein embolization (PVE) has been widely applied before extended hepatectomy; however, its clinical utility for patients with biliary cancer has not been fully addressed. METHODS: Between 1991 and 2010, 494 patients with cholangiocarcinoma (n = 353) or gallbladder cancer (n = 141) underwent PVE before extended hepatectomy. PVE was performed by a transhepatic ipsilateral approach using fibrin glue or absolute ethanol with steel coils. Surgical outcomes of this cohort were retrospectively reviewed. RESULTS: PVE-related complications requiring interventions were found in 3 (0.6%) of the 494 patients; no patient died of these complications. Among the 494 patients, 122 (24.7%) did not undergo subsequent hepatectomy. The unresectability rate was significantly higher in patients with gallbladder cancer than in those with cholangiocarcinoma [43.2% (61/141) and 17.3% (61/353), respectively, p < 0.001]. The remaining 372 patients underwent hepatectomy, and 24 (6.5%) died of postoperative complications [13 of 80 (16.3%) with gallbladder cancer vs. 11 of 292 (3.8%) with cholangiocarcinoma, p < 0.05]. The overall survival for patients with cholangiocarcinoma was significantly better than that for patients with gallbladder cancer, where the 5-year survival rate was 39 and 23%, respectively (p < 0.001). Thirty-six patients with cholangiocarcinoma and 10 patients with gallbladder cancer survived more than 5 years after extended surgery. CONCLUSION: PVE can be performed safely in patients with cholestatic liver, and it has a potential benefit for patients with advanced biliary cancer who are to undergo extended, difficult hepatectomy.

HMGA2 promotes the migration and invasion of gallbladder cancer cells and HMGA2 knockdown inhibits angiogenesis via targeting VEGFA.

The high mobility group AT­hook 2 (HMGA2) protein has been found to be upregulated in the majority of tumor types and is associated with a poor prognosis. Previous studies have suggested the oncogenic role of HMGA2 in gallbladder cancer (GBC). The present study aimed to investigate the effects of HMGA2 on the invasion, migration and angiogenesis of GBC cells. To achieve this aim, HMGA2 was overexpressed or silenced in the GBC cell line, EH­GB1, and then the proliferation, migration, invasion and epithelial­mesenchymal transition (EMT) abilities of EH­GB1 cells were investigated using Cell Counting Kit­8, wound healing, Transwell and western blotting assays. In addition, the expression levels of VEGFA were determined in EH­GB1 cells using western blotting and reverse transcription­quantitative PCR following HMGA2 overexpression or silencing. Furthermore, HMGA2­silenced EH­GB1 cells were transfected with VEGFA overexpression plasmids to evaluate the tube formation ability of HUVECs using tube formation assay. The results demonstrated that HMGA2 silencing inhibited GBC cell proliferation, migration, invasion and EMT, as evidenced by the downregulated expression of Ki67, proliferating cell nuclear antigen, MMP2, MMP9, N­cadherin, snail family transcriptional repressor 2 and zinc finger E­box­binding homeobox 1, and attenuated cell migration and invasion. However, the opposite results were obtained following HMGA2 overexpression. Moreover, HMGA2 knockdown and overexpression downregulated and upregulated VEGFA expression, respectively. In addition, the tube formation ability of HUVECs and the expression levels of CD31, VEGFR1 and VEGFR2 were downregulated following HMGA2 silencing. However, these effects were partially rescued by simultaneous VEGFA overexpression. In conclusion, the findings of the present study revealed that HMGA2 may promote GBC cell migration, invasion, EMT and angiogenesis. Therefore, inhibiting HMGA2 expression could be considered as a possible therapeutic approach for GBC.

Vascular evaluation using transabdominal ultrasound for gallbladder polyps.

Ultrasound (US) is a cost-effective and noninvasive procedure without radiation exposure, with real-time evaluation and high spatial resolution. Although it is useful for the detection of gallbladder (GB) polyps, including gallbladder cancer, adenoma, and benign polyps, conventional US is insufficient for differential diagnosis because it is not capable of evaluating hemodynamic information, unlike computed tomography or magnetic resonance imaging. With recent technological advances in US equipment and contrast agents, Doppler imaging and contrast-enhanced ultrasonography (CEUS) are being used to characterize GB polyps, and several reports on evaluation of the vascularity of GB polyp have been published. In this review, we aimed to report the latest developments in the hemodynamic diagnosis of GB polyps based on previous reports, with an emphasis on CEUS, and to evaluate the efficacy for differential diagnosis. The information in this article is expected to enable early diagnosis and prompt surgical treatment for gallbladder cancer.

[Expressions of VEGF-C and VEGF-D and their correlation with lymphangiogenesis and angiogenesis in gallbladder carcinoma].

OBJECTIVE: To investigate the expression of VEGF-C and VEGF-D and their correlations with lymphangiogenesis and angiogenesis in gallbladder carcinoma. METHODS: Fifty cases of gallbladder carcinoma with complete clinical and pathological data were analyzed. The expression of VEGF-C and -D, D2-40, CD31 was assayed by immunohistochemical staining, with 10 samples of normal gallbladder tissues away from cancer and 19 samples of chronic cholecystitis as controls, and their correlation with clinicopathological findings were analyzed retrospectively. RESULTS: Thirty-two (64.0%) of the 50 gallbladder cancers were positive for VEGF-C protein expression by immunohistochemistry and the positive rate of VEGF-D protein expression was 62.0% (31/50). The protein expression of VEGF-C and VEGF-D in tumor tissues was significantly higher than that in normal gallbladder tissues away from the tumor (P < 0.05), but no correlation with that in chronic cholecystitis (P < 0.05). The VEGF-C expression correlated with the patient age and lymph node metastasis (both P < 0.05). The VEGF-D expression only correlated with lymph node metastasis (P < 0.05). In the 50 gallbladder cancers, the MLVD was 6.9 + or - 3.6 and the MVD was 36.1 + or - 12.8. The MLVD in both VEGF-C and -D positive groups was significantly higher than that in the negative groups (P = 0.000), and the lymph node metastasis also increased. MVD in both VEGF-C and -D positive groups was higher than that in the negative groups (P < 0.05), and it was also correlated with tumor differentiation (P < 0.05). A significant positive correlation was also found between VEGF-C and VEGF-D expression (r = 0.498, P < 0.01). CONCLUSION: VEGF-C and VEGF-D are involved in the lymphangiogenesis and angiogenesis in gallbladder carcinoma, promote lymph node metastasis of the tumor, and both are important in the regulation of lymphangiogenesis and angiogenesis in this cancer. VEGF-C and VEGF-D are of clinical significance in evaluating lymph node metastatic potency and estimation of prognosis in gallbladder carcinoma.

Gallbladder cancer-associated fibroblasts promote vasculogenic mimicry formation and tumor growth in gallbladder cancer via upregulating the expression of NOX4, a poor prognosis factor, through IL-6-JAK-STAT3 signal pathway.

BACKGROUND: Cancer-associated fibroblasts (CAFs) and vasculogenic mimicry (VM) play important roles in the occurrence and development of tumors. However, the relationship between CAFs and VM formation, especially in gallbladder cancer (GBC) has not been clarified. In this study, we investigated whether gallbladder CAFs (GCAFs) can promote VM formation and tumor growth and explored the underlying molecular mechanism. METHODS: A co-culture system of human GBC cells and fibroblasts or HUVECs was established. VM formation, proliferation, invasion, migration, tube formation assays, CD31-PAS double staining, optic/electron microscopy and tumor xenograft assay were used to detect VM formation and malignant phenotypes of 3-D co-culture matrices in vitro, as well as the VM formation and tumor growth of xenografts in vivo, respectively. Microarray analysis was used to analyze gene expression profile in GCAFs/NFs and VM (+)/VM (-) in vitro. QRT-PCR, western blotting, IHC and CIF were used to detected NOX4 expression in GCAFs/NFs, 3-D culture/co-culture matrices in vitro, the xenografts in vivo and human gallbladder tissue/stroma samples. The correlation between NOX4 expression and clinicopathological and prognostic factors of GBC patients was analyzed. And, the underlying molecular mechanism of GCAFs promoting VM formation and tumor growth in GBC was explored. RESULTS: GCAFs promote VM formation and tumor growth in GBC; and the finding was confirmed by facts that GCAFs induced proliferation, invasion, migration and tube formation of GBC cells in vitro, and promoted VM formation and tumor growth of xenografts in vivo. NOX4 is highly expressed in GBC and its stroma, which is the key gene for VM formation, and is correlated with tumor aggression and survival of GBC patients. The GBC patients with high NOX4 expression in tumor cells and stroma have a poor prognosis. The underlying molecular mechanism may be related to the upregulation of NOX4 expression through paracrine IL-6 mediated IL-6/JAK/STAT3 signaling pathway. CONCLUSIONS: GCAFs promote VM formation and tumor growth in GBC via upregulating NOX4 expression through the activation of IL-6-JAK-STAT3 signal pathway. NOX4, as a VM-related gene in GBC, is overexpressed in GBC cells and GCAFs, which is related to aggression and unfavorable prognosis of GBC patients.

The prognostic value of angiogenesis by Chalkley counting in gallbladder carcinoma.

BACKGROUND/AIMS: Tumor angiogenesis has been proposed as essential to tumor growth, proliferation and metastases however, for gallbladder neoplasia, these data remain obscure. Therefore, it was performed this study with the objective of evaluating the prognostic value of angiogenesis in gallbladder cancer. METHODOLOGY: Thirty cases of gallbladder carcinoma were studied by immunohistochemical technique with antibody anti-CD105 for the identification of neoformed intratumoral vessels. The intratumoral micro vessel density was evaluated by Chalkley counting method, and researched the association of this index with the survival rate and the prognostic factors. RESULTS: It was obtained, starting from the analysis of these data, a cut off in the index of the tumor vascular density at 8.5. It was observed an association between high intratumoral micro vessel density (index above or equal to 8.5) and survival rate lower to 6 months in 84.6% of cases. CONCLUSION: The present study confirms the hypothesis that a high vascular density, appraised through the Chalkley counting method, is associated to a worse prognosis in patients of gallbladder carcinoma.

Multimedia article. Totally laparoscopic extended right hepatectomy.

BACKGROUND: This video demonstrates the relevant technical maneuvers necessary for performing a totally laparoscopic extended right hepatectomy. METHODS: The five principal steps of this procedure include mobilization of the liver, control of hepatic inflow, division of the hepatic parenchyma, control of the hepatic outflow, and removal of the specimen. RESULTS: A total of five totally laparoscopic extended right hepatectomies have been performed successfully at our institution. Our short- and long-term results have been similar to those for our open historical control subjects. No mortalities have been observed. CONCLUSION: The minimally invasive approach to hepatic resections is limited by the comfort level of the operator and not the technique itself.

[A case of unresectable advanced gallbladder cancer successfully treated by oral S-1 and hepatic arterial infusion (HAI) of low-dose CDDP therapy].

The patient was a 64-year-old woman. Oral S-1 and hepatic arterial infusion (HAI) of low-dose CDDP therapy were started for unresectable advanced gallbladder cancer associated with liver metastasis and numerous lymph node metastases. Marked regression of the liver metastasis and lymph node metastases was observed by this treatment, and upon completion of the second course they had almost completely resolved. The tumor marker values also converted to negative. We report a case in which oral S-1 and HAI of low-dose CDDP therapy was effective against advanced gallbladder cancer associated with liver metastasis and multiple lymph node metastases.

miR-143-3p targeting of ITGA6 suppresses tumour growth and angiogenesis by downregulating PLGF expression via the PI3K/AKT pathway in gallbladder carcinoma.

Gallbladder cancer (GBC) is the most common malignant tumour of the biliary track system. Angiogenesis plays a pivotal role in the development and progression of malignant tumours. miR-143-3p acts as a tumour suppressor in various cancers. Their role in GBC is however less well defined. Here we show that the expression levels of miR-143-3p were decreased in human GBC tissues compared with the non-tumour adjacent tissue (NAT) counterparts and were closely associated with overall survival. We discovered that miR-143-3p was a novel inhibitor of tumour growth and angiogenesis in vivo and in vitro. Our antibody array, ELISA and PLGF rescue analyses indicated that PLGF played an essential role in the antiangiogenic effect of miR-143-3p. Furthermore, we used miRNA target-prediction software and dual-luciferase assays to confirm that integrin α6 (ITGA6) acted as a direct target of miR-143-3p. Our ELISA and western blot analyses confirmed that the expression of PLGF was decreased via the ITGA6/PI3K/AKT pathway. In conclusion, miR-143-3p suppresses tumour angiogenesis and growth of GBC through the ITGA6/PI3K/AKT/PLGF pathways and may be a novel molecular therapeutic target for GBC.

Diagnosis of gallbladder diseases by contrast-enhanced phase-inversion harmonic ultrasonography.

We evaluated the usefulness of contrast-enhanced ultrasonography(US) for detecting and differentiating gallbladder lesions. Contrast-enhanced coded phase-inversion harmonic US was performed on 90 patients with gallbladder abnormalities. After administering Levovist, we observed the gallbladders in real time. Contrast-enhanced coded phase-inversion harmonic ultrasonography was compared with B-mode US and contrast-enhanced computer tomography (CT) with regard to the sensitivity and specificity in depicting the elevated gallbladder lesions. Furthermore, we assessed how the vascular patterns of the elevated gallbladder lesions depicted by contrast-enhanced US correlated with the diagnosis. Contrast-enhanced US efficiently discriminated true lesions from biliary sludge, unlike B-mode US. Consequently, contrast-enhanced US was more specific (100%) than B-mode US (81%), although their sensitivities were similar (98% and 96%, respectively). Contrast-enhanced US was also more sensitive that contrast-enhanced CT (98% versus 79%), although the two methods were equally sensitive (100% versus 95%). We classified the vascular patterns of the abnormalities depicted by contrast-enhanced US in the 90 cases into types 1 to 4, which represent branch-like, heterogeneous, homogeneous, and avascular patterns, respectively. All type 1 and 2 lesions were over 10 mm in size while most (88%) type 3 lesions were 10 mm or less in size. While the majority of carcinomas (86%) were type 1 or 2, three benign lesions also showed these patterns. Thus, the vascular pattern may simply reflect the size of the lesion and therefore its usefulness in diagnosing gallbladder lesions may be limited. Nevertheless, contrast-enhanced US is clearly superior to the other techniques in discriminating biliary sludge from other lesions.

[Vasculogenic mimicry in human primary gallbladder carcinoma and clinical significance thereof].

OBJECTIVE: To investigate if there is vasculogenic mimicry (VM) in human primary gallbladder carcinoma and clinical significance thereof. METHODS: Seventy-four specimens of primary gallbladder carcinoma obtained from operation underwent HE staining and double staining of CD(31) and PAS to observe the existence of VM. The correlation of pathological examination and clinical outcomes was analyzed. RESULTS: VM was seen in 10 of the 74 (13.5%) specimens. VM was not correlated with age, sex, location, diameter, differentiation degree, Nevin stage, and invasion depth of tumor, and existence of lymph node metastasis; but was associated with histological type (chi(2) = 10.241, P = 0.017), hepatic metastasis (chi(2) = 11.904, P = 0.01), and poor overall survival (chi(2) = 5.771, P = 0.016). Cox analysis showed that existence of VM, invasion depth, lymph node metastasis, hepatic metastasis, and operational method were independent risk factors of the prognosis of primary gallbladder carcinoma. CONCLUSION: VM exists in human primary gallbladder carcinoma. Those cases of human primary gallbladder carcinoma with VM have a poorer prognosis.

DUSP1 inhibits cell proliferation, metastasis and invasion and angiogenesis in gallbladder cancer.

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPK activity and is known to play a key role in tumor biology. Its function in gallbladder cancer (GBC) remains largely unknown, however. By exploring its activities in two GBC cell lines (SGC996 and GBC-SD), DUSP1 was found to inhibit GBC cell proliferation, migration and invasion. Moreover, DUSP1 inhibited GBC growth and metastasis in nude mice subcutaneously xenografted with SGC996 cells. The tumor suppression appeared to be mediated via the DUSP1-pERK/MAPK-MMP2 signal pathway. Angiogenesis was associated with the tumor metastasis in the mouse model and was impaired by DUSP1, which suppressed VEGF expression. These results suggest that DUSP1 suppresses GBC growth and metastasis by targeting the DUSP1-pERK-MMP2/VEGF axis. Identification of the DUSP1-pERK-MMP2/VEGF signals may provide new biomarkers and/or therapeutic targets to better suppress GBC metastasis in the future.

Analysis of p53 and vascular endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity.

AIM: To examine the expression of p53 and vascular endothelial growth factor (VEGF) as well as microvessel count (MVC) and to investigate the role of VEGF as an angiogenic marker and the possible role of p53 in the regulation of angiogenesis in human gallbladder carcinoma. METHODS: Surgically resected specimens of 49 gallbladder carcinomas were studied by immunohistochemical staining for p53 protein, VEGF, and factor VIII-related antigen. VEGF expression and mutant p53 expression were then correlated with Nevin stage, differentiation grade, MVC, and lymph node metastasis. RESULTS: Positive p53 protein and VEGF expressions were found in 61.2% and 63.3% of tumors, respectively. p53 and VEGF staining status was identical in 55.1% of tumors. The Nevin staging of p53- or VEGF-positive tumors was significantly later than that of negative tumors. The MVC in p53- or VEGF-positive tumors was significantly higher than that in negative tumors, and MVC in both p53- and VEGF-negative tumors was significantly lower than that in the other subgroups. CONCLUSION: Our findings suggest that p53-VEGF pathway can regulate tumor angiogenesis in human gallbladder carcinoma. Combined analysis of p53 and VEGF expression might be useful for predicting the tumor vascularity of gallbladder cancer.

[Effects of norcantharidin on angiogenesis of human gallbladder carcinoma and its anti-angiogenic mechanisms].

OBJECTIVE: To investigate the effects of norcantharidin (NCTD) on angiogenesis of human gallbladder carcinoma and its anti-angiogenic mechanisms. METHODS: Human gallbladder carcinoma cells of the line GBC-SD were cultured. BALB/c nude mice were inoculated subcutaneously with the GBC-SD cells and then randomly divided into 6 groups: NCTD group, injected intraperitoneally with 1/5 of the LD(50) of NCTD twice a week for 6 weeks; 5-fluorouracil (5-FU) group, injected intraperitoneally with 1/5 of the LD(50) of 5-FU twice a week for 6 weeks; endostatin (ES) group, intraperitoneally with ES; NCTD + 5-FU group, injected intraperitoneally with 1/5 of the LD(50) of NCTD and 1/5 of the LD(50) of 5-FU twice a week for 6 weeks; NCTD + ES group, injected intraperitoneally with 1/5 of the LD(50) of NCTD and ES twice a week for 6 weeks; and normal saline (NS) group (control group), injected with NS. The mice were killed in the 7th week. The tumors were taken out to measure their volumes and undergo microscopy. SABC method of immunohistochemistry was used to measure the microvessel density (MVD) and the protein expression of the angiogenesis-related factors: proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), angiopoietin (Ang)-2, thrombospondin (TSP), and tissue inhibitor of metalloprotease (TIMP)(2). Suspension of single tumor cell was prepared to examine the cell apoptosis by flow cytometry. RT-PCR was used to examine the mRNA expression of PCNA, VEGF, Ang-2, TSP, and TIMP2. RESULTS: (1) The MVD of the NCTD group was 4.12 +/- 1.4, significantly lower than those of the 5-FU group (15.8 +/- 5.9) and control group (17.6 +/- 3.2) (both P < 0.01), but not significantly different from those of the NCTD + 5-FU group (3.8 +/- 1.7), ES group (4.5 +/- 2.1), and NCTD + ES group (2.9 +/- 1.5) (all P > 0.05). The mice treated with NCTD showed significantly smaller tumor volume, lower PCNA protein expression, higher apoptotic rate, and higher PCNA/apoptosis ratio (P < 0.05 or P < 0.01), and significant correlation between MVD and tumor volume and between MVD and PCNA/apoptosis ratio (both P < 0.05). (2) The protein expression of VEGF and of Ang-2 of the NCTD group were both significantly lower than those of the control and 5-FU groups (all P < 0.01), however, not significantly different from those of the ES, NCTD + 5-FU, and NCTD + ES groups; and the protein expression of TSP and of TIMP2 of the NCTD group were both significantly higher than those of the control and 5-FU groups (all P < 0.01), however, not significantly different from those of the ES, NCTD + 5-FU, and NCTD + ES groups. MVD was positively correlated with VEGF and Ang-2 expression and negatively correlated with the expression of TSP and TIMP2 (all P < 0.05). (3) In comparison with the control group, the mRNA expression of VEGF and of Ang-2 of the tumor cells of the NCTD group were both significantly lower and the mRNA expression of TIMP2 was significantly higher. CONCLUSION: NCTD down-regulates the expression of the angiogenic factors, such as VEF|GF and Ang-2, and up-regulates the expression of the anti-angiogenic factors, such as TDP and TIMP2, thus inhibiting the angiogenesis in tumor, such as human gallbladder carcinoma, and further inhibiting the growth of tumor.

Cyclooxygenase-2 expression determines neo-angiogenesis in gallbladder carcinomas.

Neo-angiogenesis may have an important role in the poor prognosis of gallbladder carcinoma. An enhanced expression of COX-2 was found in precancerous lesions and in gallbladder carcinoma, likely to be involved in carcinogenesis as well as in angiogenesis. To study the relationships between the COX-2 expression and degree of vascularization, as well as to evaluate their role in the prognosis of patients with gallbladder carcinoma. 27 cases of gallbladder adenocarcinoma were included, classified grading I-III according the WHO classification. The COX-2 and endothelial antigen CD105 expressions were assessed immunohistochemically. COX-2 expression was evaluated according to the percentage and staining intensity of positive cells into "COX-2 positive" and "COX-2 negative" groups. In order to assess tumor microvessel density (MVD), CD105 positively stained microvessels were counted for each specimen in predominantly vascular areas (hot spots) at 200 x magnification. The MVD ranged from 9 to 46 microvessels/field. 15 tumors belonged to the hypervascular group (MVD > or = 25) and 12 to the hypovascular group. There were 16 (59.2%) COX-2 positive cases. There was difference in the degree of angiogenesis between COX-2 positive vs. COX-2 negative group: 11 (68.8%) out of 16 "COX-2 positive" tumors were hypervascular, in comparison with just 4 (36.4%) of "COX-2 negative" tumors. Our data show that the MVD corresponds to the COX-2 overexpression in gallbladder carcinomas. Augmented tumor neovascularization induced by COX-2 might be responsible for the poor prognosis in gallbladder carcinoma patients.

Cyclooxygenase-2 promotes angiogenesis by increasing vascular endothelial growth factor and predicts prognosis in gallbladder carcinoma.

AIM: To investigate the relationships between the expression of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and the degree of vascularization, clinicopathologic feature, survival time of patients with gallbladder carcinomas. METHODS: Sixty-four gallbladder carcinoma specimens were evaluated for COX-2, VEGF expression by immunohistochemical methods. Microvessel counts (MVC) were determined using CD(34). The relationships between COX-2, VEGF expression, CD(34)-stained MVC, clinicopathologic features and survival time were analyzed. The correlations between COX-2 and VEGF expression, CD(34)-stained MVC were also investigated. RESULTS: COX-2, VEGF immunoreactivity were observed in 71.9% (46/64) and 54.7% (35/64) specimens, respectively. The average MVC in 64 cases of gallbladder carcinoma was 57+/-14 per high power vision field. The status of MVC was closely correlated with Nevin staging, tumor differentiation and lymph node metastasis (P<0.01, 0.002, and 0.003, 0.000, respectively). Increased VEGF expression was significantly correlated with tumor differentiation (poorly and moderately>well differentiated, P<0.05, P = 0.016). Clinical stages had no relation with the expression of VEGF (P>0.05, P = 0.612). There was a positive correlation between COX-2 expression and clinical stages. The positive rate of COX-2 was higher in cases of Nevin stages S(4)-S(5) (81.8%) than in those of Nevin stages S(1)-S(3) (50.0%) with a statistical significance (P<0.01, P = 0.009). The expression of COX-2 did not vary with differentiation (P>0.05, P = 0.067). Statistically significant differences were also observed according to lymph node metastasis, COX-2 expression and VEGF expression (P<0.01, 0.000, and 0.001, respectively). There was no relation between VEGF, COX-2 expression, MVC and the age and sex of patients. MVC and VEGF positive rate in the COX-2 positive gallbladder carcinoma tissue was higher than that in the COX-2 negative tissue (P<0.05, 0.000, and 0.032, respectively). Patients with VEGF, COX-2 positive tumors had a significantly shorter survival time than those with negative tumors (P<0.05, 0.004, 0.01, respectively). CONCLUSION: Augmented tumor neovascularization induced by VEGF may be one of the several effects of COX-2 responsible for poor prognosis of human gallbladder carcinoma. COX-2 inhibitor, either in combination therapy with other agents, or for chemoprevention, may be effective via suppression of angiogenesis in this fatal disease.