A: VEGF isoform mRNA expression was examined by RT-PCR analysis of wound mRNA from day 3, day 5, and day 7 wounds. processes such as embryonic development, reproduction, wound healing, cancer, arthritis, retinopathies, and psoriasis. 1,2 Vascular endothelial growth factor (VEGF), a potent Remodelin Hydrobromide proangiogenic factor, has been extensively studied as a mediator of angiogenesis. VEGF is expressed in high levels and plays a functional role during embryonic development, as the absence of a single VEGF allele in embryos causes embryonic Remodelin Hydrobromide lethality and abnormal vasculogenesis. 3-5 VEGF is also critical in angiogenic processes of the adult, where antibody blockade of VEGF leads to a significant decrease in angiogenic activity in wound fluids and tumors. 6-10 The three soluble isoforms of VEGF, VEGF121, VEGF145, and VEGF165, induce angiogenesis through the binding of cell surface JAG2 receptors, VEGFR1 and VEGFR2, which are expressed on the endothelial cells of sprouting vessels. VEGFR2 is capable of binding all three isoforms of VEGF, whereas VEGFR1 binds only VEGF121 and VEGF165. 11 Both VEGFR1 and VEGFR2 are functionally active during angiogenesis. 12-19 Recently, a third VEGF receptor, neuropilin-1, has been identified. Neuropilin-1 was originally characterized as a semaphorin receptor that is important for guidance of developing nerves. 20,21 Neuropilin-1 also plays a role in vasculogenesis as neuropilin-1-null mice are embryonic lethal and exhibit cardiovascular defects. 22 Recent studies have shown that expression of neuropilin-1 on endothelial cells enhances the biological activity of VEGFR2 in response to the VEGF165 isoform. 23,24 This finding suggests that neuropilin-1 is a co-receptor for VEGF, and has led to the theory that neuropilin-1 is involved in VEGF-mediated angiogenesis for 18 hours. For each total RNA preparation, four to five wounds were pooled and yielded 32 to 130 g of RNA. Northern analysis was performed Remodelin Hydrobromide by electrophoresis of 10 g of total RNA per lane through 0.8% agarose, 2 mol/L formaldehyde gels in 20 mmol/L MOPS buffer, pH 7.0. Gels were blotted onto Gene Screen Plus (DuPont-NEN, Wilmington, DE) and the membrane hybridized according to the manufacturers directions. The following templates were created for probe generation: a 760-bp reverse transcriptase-polymerase chain reaction (RT-PCR) product of the N-terminal a2 domain of neuropilin-1 was cloned into pPCR-Script Amp SK(+) (Stratagene, La Jolla, CA). A 502-bp RT-PCR product of VEGFR-1 was cloned into pPCR-Script Amp SK+ (Stratagene). A 960-bp RT-PCR fragment of VEGFR-2 was cloned into pBluescript KS+ (Stratagene). VEGFR1, VEGFR2, and neuropilin-1-specific probes were made using the RadPrime Labeling System (Life Technologies, Inc., Gaithersburg, MD) to a specific activity of at least 10 8 cpm/g. GAPDH expression levels were used for normalization. Neuropilin-1 Antiserum Production and Purification Polyclonal anti-neuropilin-1 antibodies were generated by immunizing a rabbit with a histidine-tagged neuropilin-1 protein that was produced in the BL21(DE3)pLysS strain of and isolated as previously described. 21 Rabbits were immunized with 600 g of the protein in 0.6 ml of complete Freunds adjuvant and were boosted every 2 weeks with 300 g of the protein in 0.5 ml of incomplete Freunds adjuvant. Serum Remodelin Hydrobromide was collected and purified by protein A-Sepharose chromatography to obtain the IgG fraction. The amount of rabbit IgG was determined by bicinchoninic acid protein assay (Pierce Chemical Company, Rockford, IL). By Western blot analysis, the anti-neuropilin-1 antibodies recognized the MAM fragment of neuropilin-1 as a single 40-kd band (data not shown). The ability of the anti-neuropilin-1 antibodies to block the functional activity of neuropilin-1 receptors on neurons has been previously described. 21 Immunohistochemistry Ten-m sections from frozen embedded tissues were prepared for immunohistochemical analysis of PECAM-1 and neuropilin-1 expression. The PECAM-1 analysis detects both progenitor and differentiated endothelial cells, as both populations express CD31. 30,31 All incubations and washes were performed at room temperature. Sections were fixed in acetone for 30 minutes. After three 3-minute washes in phosphate-buffered saline (PBS), pH 7.4, sections were treated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. The slides were washed in PBS, pH 7.4, and blocked with 1:10 dilution of normal mouse serum (Sigma Chemical Company, St. Louis, MO) in PBS, pH 7.4, for 30 minutes. For PECAM-1 staining, sections were incubated in 1.0 g/ml of MEC13.3 rat anti-mouse PECAM-1 antibody (anti-CD31; Pharmingen International, San Diego, CA) in PBS, pH 7.4. For the detection of neuropilin-1, sections were incubated in 93 ng/ml of purified rabbit IgG from the anti-neuropilin-1 antiserum. After a 30-minute incubation with either primary antibody, the slides were washed for 3 minutes, three times, in.