A unique protein kinase C-dependent pathway for tissue factor downregulation in pericytes.

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11 h to 4.5 h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinase C (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes.

Feasibility of using gene expression analysis to study canine soft tissue sarcomas.

The prognosis given for canine soft tissue sarcomas (STSs) is based primarily on histopathologic grade. The decision to administer adjuvant chemotherapy is difficult since less than half of patients with high-grade STSs develop metastatic disease. We hypothesize that there is a gene signature that will improve our ability to predict development of metastatic disease in STS patients. The objective of this study was to determine the feasibility of using cDNA microarray and quantitative real-time PCR (qRT-PCR) analysis to determine gene expression patterns in metastatic versus nonmetastatic canine STSs, given the inherent heterogeneity of this group of tumors. Five STSs from dogs with metastatic disease were evaluated in comparison to eight STSs from dogs without metastasis. Tumor RNA was extracted, processed, and labeled for application to the Affymetrix Canine Genechip 2.0 Array. Array fluorescence was normalized using D-Chip software and data analysis was performed with JMP/Genomics. Differential gene expression was validated using qRT-PCR. Over 200 genes were differentially expressed at a false discovery rate of 5%. Differential gene expression was validated for five genes upregulated in metastatic tumors. Quantitative RT-PCR confirmed increased relative expression of all five genes of interest in the metastatic STSs. Our results demonstrate that microarray and qRT-PCR are feasible methods for comparing gene signatures in canine STSs. Further evaluation of the differences between gene expression in metastatic STSs and in nonmetastatic STSs is likely to identify genes that are important in the development of metastatic disease and improve our ability to prognosticate for individual patients.

The effect of darbepoetin alfa on growth, oxygenation and radioresponsiveness of a breast adenocarcinoma.

Tumor hypoxia is associated with poor clinical outcome in a variety of tumors, including cervical, head/neck and breast cancer. Administration of erythropoietic factors has been suggested as a means of improving tumor oxygenation (pO2). This study randomized rats to treatment with low-dose or high-dose darbepoetin alfa or a placebo to determine the effect of darbepoetin alfa on the pO2, growth and response to radiation therapy of R3230 mammary adenocarcinoma. Rats received 3 microg/kg (high dose) or 0.2 microg/kg (low dose) darbepoetin alfa or placebo for eight doses prior to either (1) pO2 measurement and pimonidazole staining or (2) irradiation or sham irradiation on post-transplant day 20. In the animals randomized to radiation treatment, placebo or darbepoetin alfa administration at a reduced dose was continued for 9 weeks or until the tumor diameter exceeded 15 mm (defined as failure for survival analysis). Treatment with high-dose and low-dose darbepoetin alfa produced hematocrits of 68 and 56% compared to 44 and 45% in their respective control groups (both P < 10(-5)). At 18 days post-transplant, tumor volume was not different for either darbepoetin alfa group compared to the placebo group. Tumor oxygenation, as measured by the fraction of pO2 measurement <10 mmHg and the intensity of pimonidazole staining, was significantly improved in the high-dose group (P = 0.046 and 0.03, respectively, compared with controls) but not in the low-dose group. Growth delay curves after irradiation did not differ significantly for high- or low-dose darbepoetin alfa compared to placebo. In this nonanemic animal model of mammary adenocarcinoma, darbepoetin alfa does not significantly alter tumor growth or radioresponsiveness, even though it improves oxygenation when administered at high doses.

Oxygen regulation of tumor perfusion by S-nitrosohemoglobin reveals a pressor activity of nitric oxide.

In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). It has been shown that SNO-Hb behaves as a nitric oxide (NO) donor at low oxygen tensions. This property, in combination with oxygen transport capacity, suggests that SNO-Hb may have unique potential to reoxygenate hypoxic tissues. The present study was designed to test the idea that the allosteric properties of SNO-Hb could be manipulated to enhance oxygen delivery in a hypoxic tumor. Using Laser Doppler flowmetry, we showed that SNO-Hb infusion to animals breathing 21% O2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO2 (100% O2) slowed the release of NO bioactivity from SNO-Hb (ie, prolonged the plasma half-life of the SNO in Hb), preserved tumor perfusion, and raised the blood pressure. In contrast, native Hb reduced both tumor perfusion and heart rate independently of the oxygen concentration of the inhaled gas, and did not elicit hypertensive effects. Window chamber (to image tumor arteriolar reactivity in vivo) and hemodynamic measurements indicated that the preservation of tissue perfusion by micromolar concentrations of SNO-Hb is a composite effect created by reduced peripheral vascular resistance and direct inhibition of the baroreceptor reflex, leading to increased blood pressure. Overall, these results indicate that the properties of SNO-Hb are attributable to allosteric control of NO release by oxygen in central as well as peripheral issues.

The relationship between serum vascular endothelial growth factor, persistent disease, and survival at second-look laparotomy in ovarian cancer.

OBJECTIVE: To assess if the angiogenic factors vascular endothelial growth factor (VEGF) and D-dimer are predictive of persistent disease, early relapse, and survival in patients with ovarian cancer who achieve a complete clinical remission after first-line chemotherapy. METHODS: Serum levels of VEGF and D-dimer were assessed by ELISA in 62 patients who completed first-line chemotherapy and underwent second-look laparotomy at Duke University Medical Center. Cox Proportional Hazards Modeling was utilized to determine if VEGF and/or D-dimer levels could predict disease-free and overall survival. The Kaplan-Meier method was used to estimate median survival. The Wilcoxon test was used to determine if a significant difference existed in median VEGF and D-dimer levels between patients with positive and negative second-look operations. RESULTS: Forty (65%) of the 62 women who underwent second-look laparotomy had persistent disease. The median VEGF levels were 264 pg/ml (range 109-896 pg/ml) in the group with negative second looks compared to 390 pg/ml (range 99-1011 pg/ml) in those with positive second-looks (P = 0.1). High levels of VEGF were marginally associated with the presence of persistent (P = 0.10) and gross (P = 0.07) disease at the time of second look laparotomy. After adjusting for CA125, women with high VEGF serum levels had a worse overall survival (P = 0.004). CONCLUSIONS: This study suggests that serum VEGF may be a clinically important marker for persistent disease and is predictive of survival in ovarian cancer patients after first-line chemotherapy.

Human recombinant erythropoietin significantly improves tumor oxygenation independent of its effects on hemoglobin.

Tumor oxygenation is known to be an important predictive/prognostic marker in a variety of tumors, including cervix, head/neck, sarcoma, non-small cell of the lung, and breast. Tumor oxygenation is influenced by many interactions, including oxygen delivery (angiogenesis, permeability, and HgB) and consumption (metabolic and growth rates). This study randomized 30 nonanemic, female Fischer 344 rats into three treatment arms to examine the effects of recombinant human erythropoietin (EPO) on R3230 rodent mammary carcinoma oxygenation. The three treatment arms were: (a) placebo; (b) EPO after tumor implantation (2000 units/kg/SQdose, M/W/F for six doses); and (c) EPO before tumor implantation (2000 units/kg/SQdose, M/W/F for six doses). Tumors were implanted in the hindflank, and in vivo oxygenation was measured at day 22 after implantation using the Oxylite system (Oxford Optronix, Oxford, England). An average of 180 measurements/animal were performed. On day 22, median tumor volume was 399 mm(3) (range: 65-950 mm(3)), and no differences in tumor volume were seen between treatment arms. Mean hematocrit was equal between arms at therapy initiation but were significantly higher for both arms receiving EPO at day 22 (placebo versus Arm B versus Arm C; Wilcoxon P = 0.052). EPO-treated tumors had significantly less hypoxic measurements when compared with either the placebo or those receiving EPO before implantation. These data confirm that tumor oxygenation in nonanemic individuals may be improved through the administration of EPO, and this improvement appears to be independent of HgB effects.