Wogonoside alleviates high glucose-induced dysfunction of retinal microvascular endothelial cells and diabetic retinopathy in rats by up-regulating SIRT1 / 南方医科大学学报

OBJECTIVE@#To investigate the effects of wogonoside on high glucose-induced dysfunction of human retinal microvascular endothelial cells (hRMECs) and streptozotocin (STZ)-induced diabetic retinopathy in rats and explore the underlying molecular mechanism.@*METHODS@#HRMECs in routine culture were treated with 25 mmol/L mannitol or exposed to high glucose (30 mmol/L glucose) and treatment with 10, 20, 30, 40 μmol/L wogonoside. CCK-8 assay and Transwell assay were used to examine cell proliferation and migration, and the changes in tube formation and monolayer cell membrane permeability were tested. ROS, NO and GSH-ST kits were used to evaluate oxidative stress levels in the cells. The expressions of IL-1β and IL-6 in the cells were examined with qRT-PCR and ELISA, and the protein expressions of VEGF, HIF-1α and SIRT1 were detected using Western blotting. We also tested the effect of wogonoside on retinal injury and expressions of HIF-1α, ROS, VEGF, TNF-α, IL-1β, IL-6 and SIRT1 proteins in rat models of STZ-induced diabetic retinopathy.@*RESULTS@#High glucose exposure caused abnormal proliferation and migration, promoted angiogenesis, increased membrane permeability (P < 0.05), and induced inflammation and oxidative stress in hRMECs (P < 0.05). Wogonoside treatment concentration-dependently inhibited high glucose-induced changes in hRMECs. High glucose exposure significantly lowered the expression of SIRT1 in hRMECs, which was partially reversed by wogonoside (30 μmol/L) treatment; interference of SIRT1 obviously attenuated the inhibitory effects of wogonoside against high glucose-induced changes in proliferation, migration, angiogenesis, membrane permeability, inflammation and oxidative stress in hRMECs. In rat models of STZ-induced diabetic retinopathy, wogonoside effectively suppressed retinal thickening (P < 0.05), alleviated STZ-induced retinal injury, and increased the expression of SIRT1 in the retinal tissues (P < 0.001).@*CONCLUSION@#Wogonoside alleviates retinal damage caused by diabetic retinopathy by up-regulating SIRT1 expression.

Agkistrodon halys venom antitumor component-I inhibits vasculogenic mimicry in triple-negative breast cancer cells in vitro by down-regulating MMP2 / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of agkistrodon halys venom antitumor component-I (AHVAC-I) on vasculogenic mimicry (VM) formation in triple-negative breast cancer MDA-MB-231 cells and explore its possible mechanism.@*METHODS@#CCK8 assay was used to determine the optimal concentration of AHVAC-I for cell treatment based on its halfinhibitory concentration (IC50). MDA-MB-231 cells were treated with different concentrations of AHVAC-I or 5-Fu, and the changes in vasomimetic capacity of the cells were examined using Matrigel assay. The expression levels of matrix metalloproteinase-2 (MMP2) and MMP9 in the treated cells were detected using quantitative PCR and Western blotting.@*RESULTS@#Compared with the control treatment with culture medium, treatment with 5, 10 and 20 μg/mL AHVAC-I significantly reduced vasomimetic ability of MDA-MB-231 cells in a dose-dependent manner (P < 0.01). MMP2 supplementation obviously restored the vasomimetic ability of the cells inhibited by AHVAC-I.@*CONCLUSION@#AHVAC-I inhibits VM formation in triplenegative breast cancer cells in vitro by down-regulating MMP2 production.

JAG1 promotes migration, invasion, and adhesion of triple-negative breast cancer cells by promoting angiogenesis / 南方医科大学学报

OBJECTIVE@#To investigate the effect of JAG1 on the malignant phenotype of triple-negative breast cancer (TNBC) and its role in angiogenesis in breast cancer microenvironment.@*METHODS@#The expressions of Notch molecules were detected in human TNBC 231 and 231B cells using RT-qPCR. Five female nude mice were inoculated with 231 cells and another 5 with 231B cells into the mammary fat pads, and 4-6 weeks later, the tumors were collected for immunohistochemical and immunofluorescence tests. 231 cells and 231B cells were treated with recombinant JAG (rJAG) protein and DAPT, respectively, and changes in their malignant phenotypes were assessed using CCK-8 assay, Hoechst 33258 staining, wound healing assay, Transwell chamber assay and endothelial cell adhesion assay. Western blotting was used to detect the changes in the expressions of proteins related with the malignant phenotypes of 231 and 231B cells. The effects of conditioned medium (CM) derived from untreated 231 and 231 B cells, rJAG1-treated 231 cells and DAPT-treated 231B cells on proliferation and tube formation ability of cultured human umbilical vein endothelial cells (HUVECs) were evaluated using CCK-8 assay and tube-forming assay.@*RESULTS@#The expression of JAG1 was higher in 231B cells than in 231 cells (P < 0.05). Tumor 231B showed higher expression of VEGFA and CD31. Compared with 231-Blank group, the migration, invasion and adhesion of 231 cells in 231-rJAG1 were significantly enhanced (P < 0.05). Protein levels of Twist1 and Snail increased (P < 0.01), anti-apoptotic protein Bcl-2 increased (P < 0.05), while DAPT inhibited the related phenomena and indicators of 231B. The 231-rJAG1-CM increased the cell number and tubule number of HUVEC (P < 0.05).@*CONCLUSION@#JAG1 may affect the malignant phenotype of TNBC and promote angiogenesis in the tumor microenvironment.

Effect of Jianpi Huogu Formula on function damage of vascular endothelial cells induced by glucocorticoid / 中国中药杂志

This study aimed to observe the intervention effect of Jianpi Huogu Formula(JPHGF) on the functional damage of vascular endothelial cells caused by glucocorticoid, and explore its action mechanism from the PI3 K/Akt and mitogen activated protein kinase(MAPK) signaling pathways. The extracted thoracic aorta ring of normal SD rats were intervened first with vascularendothelial growth factor(VEGF, 20 μg·L-1) and/or sodium succinate(MPS, 0. 04 g·L-1) in vitro and then with JPHGF(8, 16, and 32 μg·L-1) for five mcontinuous ethylpdays, rednisolofollowed nebythe statistics of the number, length, and area of microvessels budding fromvascular rings. In addition, the human umbilical vein endothelial cells(HUVECs) induced by VEGF(20 μg·L-1) were added with MPS(0. 04 g·L-1) and then with JPHGF(8, 16, and 32 μg·L-1) for observing the migration, invasion, and luminal formation abilities of HUVECs in the migration, invasion and luminal formation experiments. The protein expression levels of PI3 K, p-Akt, p-JN K, and p-ERK in HUVECs were assayed by Western blot. The results showed that JPHGF dose-dependently improved the num-ber,length, and area of microvessels in MPS-induced rat thoracic aortic ring, reversed the migration, invasion and lumen formation abiliti es of HUVECs reduced by MPS, and up-regulated the protein expression levels of PI3 K, p-Akt, and p-JNK in HUVECs. All thesehave suggested that JPHGF exerts the protective effect against hormone-induced damage to the angiogenesis of vascular endothelial cells by activating the PI3 K/Akt and MAPK signaling pathways, which has provided reference for exploring the mechanism of JPHGF in treating s teroid-induced avascular necrosis of femoral head(SANFH) and also the experimental evidence for enriching the scientific connotationof spleen-invigorating and blood-activating therapy.

Assessment of Mean Vessel Density and Angiogenesis in Endometrial Carcinomas.

Objective: To evaluate the role of angiogenesis in tumor growth by the assessment of mean vessel density and to quantify angiogenesis as an important variable in endometrial cancers. Material and Methods: 53 cases of endometrial malignancies (epithelial tumors-36 cases and metastatic tumors-17 cases), were analysed for histological types, grades and features like depth of invasion and vascular invasion. Microvessel counts were performed by examining the microvessels thoroughly in terms of count, morphology and density after staining the tissues by hematoxylin & eosin stain, reticulin and immunostain (Antifactor VIII Ag). Results: On H&E stain - Microvessel density (MVD) in endometrial malignancy ranged from 3.0 - 13.5 and mean MVD was 8.78. On Reticulin stain - MVD ranged from 3.5 - 15.2 and mean MVD was 9.76. Antifactor VIII sections showed very small microvessels or even single endothelial cells with the highest total counts and the MVD ranged from 6.5- 16.8 with Mean MVD of 11.7. The counts increased with the grade of the tumor in the absence of necrosis or haemorrhage. MVD counts also increased with the stage, being 8.12 in Stage I disease, 8.65 in Stage II and 10.8 in stage III disease. Atypical hyperplasia was found to be associated with epithelial tumors in 8 cases, making it a significant finding. Conclusion: Role of angiogenesis assumes greater significance with increasing severity of lesions, higher grade and stage of the tumor and seems to have an important diagnostic and prognostic significance.

Antiangiogenesis therapy: an update after the first decade

Angiogenesis is a complex biological phenomenon that forms new blood vessels from the pre-existing vasculature. Aberrant angiogenesis has been implicated in a variety of diseases such as cancer, atherosclerosis, arthritis, obesity, pulmonary hypertension, diabetic retinopathy, and age-related macular degeneration. These conditions collectively affect nearly 10% of the global population. Much effort has focused on identifying new therapeutic agents that inhibit pathological angiogenesis since 1971, when Judah Folkman published the hypothesis that tumor growth is angiogenesis-dependent and that its inhibition may be therapeutic. In 2004, the U.S. Food and Drug Administration approved the first antiangiogenic drug for the treatment of metastatic colon cancer, bevacizumab (Avastin, Genentech). This drug is a humanized monoclonal antibody that neutralizes the vascular endothelial growth factor. It is used in combination with chemotherapy, and its use began the era of antiangiogenesis therapy. Several new therapeutic agents have been added to the list of approved drugs, and clinical trials of new therapeutic options and antiangiogenic agents are ongoing. This review describes the progress made in the first decade of antiangiogenesis therapy, and addresses both validated and possible targets for future drug development.

Tratamento da DMRI exsudativa: revisão das drogas antiangiogênicas / Wet-amd treatment: a review in the anti-vegf drugs

Degeneração Macular Relacionada à Idade (DMRI) exsudativa é a principal causa de perda visual severa em indivíduos acima de 50 anos nos países desenvolvidos. O fator de crescimento endotelial (VEGF) é considerado um dos mais importantes reguladores da angiogênese e da permeabilidade vascular . Drogas com atividade antiVEGF tem se mostrado eficaz em preservar ou melhorar a acuidade visual (AV) ao inibir a permeabilidade vascular e o crescimento neovascular nos pacientes tratados. Este artigo de revisão descreve o atual uso terapêutico das medicações antiVEGF para DMRI exsudativa e fornece uma visão geral do futuro da terapia antiangiogênica.

Neovascular age-related macular degeneration is the leading cause of severe, irreversible vision loss in individuals over 50 years in developed countries. Vascular endothelial growth factor (VEGF) has been shown to play a role in the regulation of choroidal neovascularization and vascular permeability. Anti-VEGF drugs have been shown to preserve or improve visual acuity by inhibiting vascular permeability and arresting the growth of neovascularization in the vast majority of treated patients. This review describes the current literature on the use of this therapeutic approach in the management of neovascular AMD and gives an overview of the future directions.

Total antioxidant capacity in Eales’ disease, uveitis & cataract.

Background & objectives: The human system possesses antioxidants that act harmoniously to neutralize the harmful oxidants. This study was aimed to evaluate the serum total antioxidant capacity (TAC) as a single parameter in Eales’ disease (ED) and in an acute inflammatory condition such as uveitis and in cataract which is chronic, compared to healthy controls. Methods: The TAC assay was done spectrophotometrically in the serum of Eales’ disease cases (n=20) as well as in other ocular pathologies involving oxidative stress namely, uveitis and cataract (n=20 each). The oxidative stress measured in terms of TBARS, was correlated with the TAC. Individual antioxidants namely vitamin C, E and glutathione were also estimated and correlated with TAC. Results: TAC was found to be significantly lower in Eales’ disease with active vasculitis (0.28 ± 0.09 mM, P<0.001), Eales’ disease with healed vasculitis (0.67 ± 0.09 mM), uveitis (0.46 ± 0.09 mM, P<0.001) and cataract (0.53 ± 0.1 mM, P=0.001) compared to the healthy controls, with a TAC level of 0.77 ± 0.09 mM. The TAC was found to correlate positively with vitamin E levels (P=0.05), GSH (P=0.02) but not with vitamin C, as seen in ED cases. In ED cases supplemented with vitamin E and C, there was a significant increase in the TAC level (P=0.02). Interpretation & conclusions: The TAC measurement provided a comprehensive assay for establishing a link between the antioxidant capacity and the risk of disease as well as monitoring antioxidant therapy. This method is a good substitute for assay of individual antioxidants as it clearly gives the status of the oxidative stress in the disease process.

Analysis of angiogenic factors and cyclooxygenase-2 expression in cartilaginous tumors: clinical and histological correlation

OBJECTIVES: To study the role of angiogenesis and cyclooxygenase-2 expression in cartilaginous tumors and correlate these factors with prognosis. INTRODUCTION: For chondrosarcoma, the histological grade is the current standard for predicting tumor outcome. However, a low-grade chondrosarcoma can follow an aggressive course-as monitored by sequential imaging techniques-even when it is histologically indistinguishable from an enchondroma. Therefore, additional tools are needed to help identify the biological potential of these tumors. The degree of angiogenesis that is induced by the tumor could assist in this task. Angiogenesis can be quantified by measuring the expression of vascular endothelial growth factor and CD34, and cyclooxygenase-2 can induce angiogenesis by stimulating the production of proangiogenic factors. METHODS: In total, 21 enchondromas and 58 conventional chondrosarcomas were studied by examining the clinical and histopathological findings in conjunction with the immunostaining markers of angiogenesis and cyclooxygenase- 2 expression. RESULTS: The significant variables that were associated with poor outcome were 1) higher-grade chondrosarcomas, 2) tumors that developed in flat bones, and 3) over-expression of CD34 (with a median count that was higher than 5.9 vessels in 5 high power fields). Moreover, CD34 expression (measured using the Chalkley method) revealed significantly higher microvessel density in flat bone chondrosarcomas. DISCUSSION: Previous studies have shown a positive correlation between Chalkley microvessel density and histological grade; however, in our sample, we found that the former is predictive of the outcome. Chondrosarcomas in flat bones have been shown to correlate with a poor prognosis. We also found that CD34 microvessel density values were significantly higher in flat-bone chondrosarcomas. This could explain-at least in part-the more aggressive biological course that is taken by these tumors. CONCLUSIONS: These results provide evidence that CD34 microvessel density in chondrosarcomas can be helpful in predicting patient outcome and may add to our understanding of chondrosarcoma pathogenesis.

Prognostic significance of microvessel density and mast cell density for the survival of Thai patients with primary colorectal cancer.

BACKGROUND: Angiogenesis has been found to be a reliable prognostic indicator for several types of malignancies. In colorectal cancer, however there has been controversy as to whether there is a correlation between this feature and the tumors' behavior. OBJECTIVE: Determine the correlation between microvessel density (MVD) and mast cell density (MCD) in order to evaluate these factors in terms of their prognostic relevance for primary colorectal carcinoma in Thai patients. MATERIAL AND METHOD: One hundred and thirty colorectal carcinoma patients diagnosed between January 2002 and December 2004 were identified. Eleven patients were excluded from the present study due to recurrence of colorectal carcinoma in eight cases whereas pathologic blocks were not found in three cases. One hundred and nineteen patients met all inclusion criteria and were included in the present study. Representative paraffin sections obtained by the tissue micro-array technique (9 x 5 arrays per slide) from areas of highest vascular density (hot spots) were prepared. Sections were immuno-stained by monoclonal anti CD 31 for microvessel and antibody mast cell tryptase for mast cell detections, respectively. Three readings at different periods of time under a microscopic examination of high power magnification were examined by a pathologist who was blinded to clinical data. The highest microvessel and mast cell counts were recorded as MVD and MCD. Patients were then divided into groups of high and low MVD and high and low MCD by median values (20.5 and 14.5, respectively). Overall survival of the patients in each group was estimated by the Kaplan-Meier Method while a multivariate Cox regression backward stepwise analysis was employed to find out independent prognostic factors. RESULTS: Significant positive correlation was found to exist between MVD and MCD in the hot spots (R = 0.697, p < 0.0001). Regarding their prognostic role, patients with tumors of low MVD (hypovascular) and low MCD (low mast cell counts) had significantly longer survival rates than those with hypervascular and high mast cell counts (p < 0.0001). The Multivariate Cox hazard showed that MVD and distance metastasis of cancer were independent poor prognostic factors to survival (p = 0.036 and p = 0.024, respectively). The patients with high MVD (hypervascular) tumors and with presence of distant metastasis had 1.9 and 2.5 times higher death rates than the corresponding hypovascular and non-metastatic groups, respectively during the period from January 2002 to September 2007. CONCLUSION: Assessment of microvessel density in the invasive front of primary colorectal carcinoma could serve as useful prognosis tool of primary colorectal carcinoma in Thai patients.

Inhibition of trichostatin A-induced antiangiogenesis by small-interfering RNA for thrombospondin-1

Expression of thrombospondin-1 (TSP-1), which is a known inhibitor of tumor growth and angiogenesis, is reciprocally regulated by positive regulators, such as VEGF. Additionally, trichostatin A (TSA) suppresses tumor progression by altering VEGF levels and VEGF-mediated signaling. Thus, understanding TSA-regulated TSP-1 expression and the effects of altered TSP-1 levels might provide insights into the mechanism of action of TSA in anti-tumorigenesis, and provide an approach to cancer therapy. Here, we examined the effect of TSA on TSP-1 expression, and the effects of TSA-induced TSP-1 on cell motility and angiogenesis, in HeLa and bovine aortic endothelial cells. TSA remarkably increased TSP-1 expression at the mRNA and protein levels, by controlling the TSP-1 promoter activity. Both TSA and exogenous TSP-1 reduced cell migration and capillary-like tube formation and these activities were confirmed by blocking TSP-1 with its neutralizing antibody and small-interfering RNA. Our results suggest that TSP-1 is a potent mediator of TSA-induced anti- angiogenesis.

Expression of PTEN and Its Correlation with Angiogenesis in Gastric Carcinoma / 대한소화기학회지

BACKGROUND/AIMS: Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a recently clarified tumor suppressor gene located in 10q23.3. Alterations of this gene are associated with tumor progression and unfavorable outcome in various human cancers. Recently, PTEN has a possible role in angiogenesis by modulating angiogenic factor including vascular endothelial growth factor (VEGF). The aim of this study was to investigate the roles of PTEN and VEGF status for angiogenesis in human gastric cancer. METHODS: We conducted an immunohistochemical investigation of PTEN and VEGF expression in 90 cases of paraffin section obtained from gastric cancer patients undergone surgical treatment. RESULTS: Negative expression of PTEN and positive expression of VEGF in gastric cancer tissues, were demonstrated in 40.0% and 77.8% of cases, respectively. However, no significant correlation was found between PTEN, VEGF expression and various clinicopathological parameters. PTEN expression did not correlate significantly with VEGF expression (p=0.301). High microvessel density (MVD) was significantly associated with lymph node metastasis and poor survival (p=0.014, 0.011, respectively). The mean MVD value of PTEN negative tumors was 90.4+/-43.0 and significantly higher than that of PTEN positive tumors (p=0.028). The mean MVD value of VEGF positive tumors was 86.4+/-36.7 and significantly higher than that of VEGF negative tumors (p=0.002). The mean MVD value of PTEN negative and VEGF positive tumors was 98.0+/-42.2, and significantly higher than those of the others. CONCLUSIONS: These results suggest that loss of PTEN expression may play a critical role in tumor progression and metastasis by stimulating tumor angiogenesis in human gastric cancer.

The Role of Angiostatin, Vascular Endothelial Growth Factor, Matrix Metalloproteinase 9 and 12 in the Angiogenesis of Hepatocellular Carcinoma / 대한간학회지

BACKGROUND/AIMS: Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. Hepatocellular carcinoma (HCC) has become a common malignant tumor worldwide. It is characterized by a high vascularity. METHODS: We studied the immunohistochemical expression of angiostatin, vascular endothelial cell growth factor (VEGF), matrix metalloproteinase (MMP)-9 and MMP-12, and the relationship between these results and the microvessel density (MVD) in 48 HCC specimens. To determine whether HCC cells express angiostatin per se, we examined the expression of angiostatin, MMP-9 and MMP-12 by Western blotting in four HCC cell lines. RESULTS: Expression of angiostatin and MMP-12 (but not MMP-9) were strongly correlated with decreased MVD in HCCs (P=0.006, P=0.038, respectively). VEGF positive tumors showed a significantly higher MVD than VEGF negative tumors (P=0.01). We divided the 48 cases into the following four groups: group A, angiostatin (+), MMP-9 or -12 (+), and VEGF (-); group B, angiostatin (-) and VEGF (-); group C, angiostatin (+), MMP-9 or -12 (+), and VEGF (+); group D, angiostatin (-) and VEGF (+). There was a significant correlation with MVD among these groups (P<0.001). Angiostatin was detected by Western blotting in 2 out of 4 HCC cell lines and was associated with plasminogen and MMP expression. CONCLUSIONS: These results indicate that angiogenesis in HCC is a complex process involving multiple factors including angiostatin, VEGF, and MMP. Our results suggest that angiostatin is generated by MMP-mediated proteolysis of plasminogen in HCC cells.

The Relationship between Microvessel Count and the Expression of Vascular Endothelial Growth Factor, p53, and K-ras in Non-Small Cell Lung Cancer

Using immunohistochemical staining, we studied the relationship between the microvessel count (MC) and the expression of K-ras, mutant p53 protein, and vascular endothelial growth factor (VEGF) in 61 surgically resected non-small cell lung cancers (NSCLC) (42 squamous cell carcinoma, 14 adenocarcinoma, 2 large cell carcinoma, 2 adenosquamous carcinoma, and 1 mucoepidermoid carcinoma). MC of the tumors with lymph node (LN) metastasis was significantly higher than that of tumors without LN metastasis (66.1+/-23.1 vs. 33.8+/-13.1, p<0.05). VEGF was positive in 54 patients (88.5%). MC was 58.1+/-25.2 (mean+/-S.D.) in a x200 field, and it was significantly higher in VEGF(+) tumors than in VEGF(-) tumors (61.4+/-23.7 vs. 32.9+/-23.8, p<0.05). VEGF expression was higher in K-ras-positive or mutant p53-positive tumors than in negative tumors (p<0.05). MC was significantly higher in K-ras(+) tumors than in K-ras(-) tumors, although it did not differ according to the level of mutant p53 protein expression. Survival did not differ with VEGF, mutant p53, or K-ras expression, or the level of MC. In conclusion, there is a flow of molecular alterations from K-ras and p53, to VEGF expression, leading to angiogenesis and ultimately lymph node metastasis. Correlations between variables in close approximation and the lack of prognostic significance of individual molecular alterations suggest that tumorigenesis and metastasis are multifactorial processes.

Angiogênese e o potencial maligno do adenocarcinoma colorretal avançado / Angiogenesis and the malignant potential of the advanced colorectal adenocarcinoma

The colorectal carcinoma is one of the most prevalent solid tumors in the word. It is the third most frequent tumor in both sexes, being preceded by lung and stomach carcinoma among men and breast and cervix carcinoma among women. In Brazil, the colorectal carcinoma is among the five more usual neoplasias, ranked fifth in mortality. In 1997, an incidence of 8980 new cases among men and 8650 new cases among women was estimated. The colorectal carcinoma has a global survival rate of 40 per cent, and a small increase has been shown in the last 40 years. The prognostic indicators in this type of cancer are the histological differentiation, the depth of invasion and the lymphatic invasion. Molecular and immunohistochemical approaches have been recently made in order to find a new prognostic indicator other than ones mentioned above. The quantitative analysis of tumor angiogenesis, defined as the growth of new capilars toward the tumor, has been shown to have clinical application in the survival and recurrence analysis. 48 patients undergoing surgery of colorectal carcinoma at The First Department of Internal Medicine, Division of Gastroenterology - Hiroshima University School of Medicine - from 1988 to 1991 were retrospectively studie. There was a significative statistical correlation between angiogenesis and the depth of tumor invasion, and it was demonstrated that the tumor spreading activity is supported by angiogenesis. A non-significative statistical association was found between the angiogenesis quantificationm, hematogenic metastasis, survival and clinical-pathological variables such as size and histological differentiation. The standardization of the immunohistochemistry and the methodology of microvascular quantification are fundamental for comparing results and using angiogenesis as a reliable prognostic indicator.