Metformin or TRAIL alone slightly induced apoptosis in these three pancreatic
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recruitment of BM progenitor cells expressing VEGFR1 and CXCR4. The results indicate that VEGFR1-TK signaling is key regulator mobilization of proangiogenic CXCR4+VEGFR1+ to the ischemic muscle and that the contribution of BM-derived CXCR4+VEGFR1+ depends on VEGFR1-TK signaling. Methods Animals and Surgery Male 8-week-old C57Bl/6 mice were obtained from Crea Japan. VEGFR1-TK knockout mice were developed on a C57Bl/6 hybrid background. The model of hind limb ischemia has been described previously. Briefly, a slit was made in the abdominal skin, permitting dissection to expose the femoral artery in the upper part of the left limb. The artery was ligated both proximally and distally with 60 silk sutures, the intervening 6 mm section was excised, and the incision was closed. The VEGF-A neutralizing antibody , VEGFR2 tyrosine kinase inhibitor ZD6474 and CXCR4 antibody were injected daily into the mice after femoral artery ligation. Mice were anesthetized by intraperitoneal injection of ketamine and xylazine throughout the experiments, and its adequacy was monitored from the disappearance of the pedal withdrawal reflex. All animal experimental procedures were approved by the Animal Experimentation and Ethics Committee of the Kitasato University School of Medicine, and were performed in accordance with the guidelines for animal experiments set down by Kitasato University School of Medicine and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The mice were maintained at a constant humidity and temperature and were kept continuously on a 12-hour light/dark cycle. All animals were provided with food and water ad libitum. At the end point of experiments, mice were sacrificed by an intraperitoneal administration of an overdose of pentobarbiturate. Mice exhibiting symptoms of infection including suppressed appetite, purulent discharge from the wound were removed from TG100 115 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19744340 the study prior to the study endpoint. Laser Doppler Imaging Blood flow to the right and left hind limbs was assessed by scanning the lower abdomen and limbs of the mice using scanning laser Doppler imaging . The ratio of blood flow in the ischemic limb to that in the control limb was calculated by dividing the integrated blood flow in an area that included the left foot pad by the integrated blood flow in an area of the same size that included the right foot pad. Blood flow measurements were assessed by scanning preoperatively, postoperatively, and on days 3, 7, 14, 21, and 28. Morphological Quantification of Blood Vessel Formation After dissection, the muscle tissues were immediately fixed with 4% paraformaldehyde in 0.1 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741728 M PBS, dehydrated in a graded series of ethanol solutions, and embedded in paraffin. Sections of paraffin-embedded tissues were mounted on glass slides, deparaffinized with xylene, and placed in cold acetone for immunostaining. Staining was performed with a Vectastain ABC kit as follows: sections were 1) incubated with diluted normal horse serum, 2) incubated overnight with diluted CD31 polyclonal antibody, 3) incubated with biotinylated anti-IgG, 4) incubated with avidin-biotin-peroxidase complex, and 5) placed in 0.02% 3,3′-diaminobenzine and 0.3% nickel ammonium sulfate in 50 mM Tris-HCl buffer. Color was developed by immersion in DAB solution containing 0.005% H2O2, and examination and photomicrography were performed with a light microscope. To avoid nonspecific staining of CD31, antibody was dil