The lymphangiogenic potency of endothelial growth factors has not been studied to date. This is partially due to the lack of in vivo lymphangiogenesis assays. We have studied the lymphatics of differentiated avian chorioallantoic membrane (CAM) using microinjection of Mercox resin, semi- and ultrathin sectioning, immunohistochemical detection of fibronectin and alpha-smooth muscle actin, and in situ hybridization with VEGFR-2 and VEGFR-3 probes. CAM is drained by lymphatic vessels which are arranged in a regular pattern. Arterioles and arteries are accompanied by a pair of interconnected lymphatics and form a plexus around bigger arteries. Veins are also associated with lymphatics, particularly larger veins, which are surrounded by a lymphatic plexus. The lymphatics are characterized by an extremely thin endothelial lining, pores, and the absence of a basal lamina. Patches of the extracellular matrix can be stained with an antibody against fibronectin. Lymphatic endothelial cells of differentiated CAM show ultrastructural features of this cell type. CAM lymphatics do not possess mediae. In contrast, the lymphatic trunks of the umbilical stalk are invested by a single but discontinuous layer of smooth muscle cells. CAM lymphatics express VEGFR-2 and VEGFR-3. Both the regular pattern and the typical structure of these lymphatics suggest that CAM is a suitable site to study the in vivo effects of potential lymphangiogenic factors. We have studied the effects of VEGF homo- and heterodimers, VEGF/PlGF heterodimers, and PlGF and VEGF-C homodimers on Day 13 CAM. All the growth factors containing at least one VEGF chain are angiogenic but do not induce lymphangiogenesis. PlGF-1 and PlGF-2 are neither angiogenic nor lymphangiogenic. VEGF-C is the first lymphangiogenic factor and seems to be highly chemoattractive for lymphatic endothelial cells. It induces proliferation of lymphatic endothelial cells and development of new lymphatic sinuses which are directed immediately beneath the chorionic epithelium. Our studies show that VEGF and VEGF-C are specific angiogenic and lymphangiogenic growth factors, respectively.

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Platelet-derived growth factor (PDGF) has a targeted activity on mesenchymal cells, but the in vivo effects of PDGF are not well understood. We have applied about 3 microg of PDGF-A and PDGF-B on the differentiated chorioallantoic membrane (CAM) of 13-day-old chick embryos. After 1-3 days, specimens were evaluated macroscopically, histologically with semi- and ultrathin sections, and immunohistologically with antibodies against smooth muscle alpha-actin (alphaSMA), desmin, and fibronectin (FN). Proliferation studies were performed according to the 5-bromo-2-deoxyuridine (BrdU)/anti-BrdU method. We did not observe effects of PDGF-A. PDGF-B induced proliferation of fibrocytes and their transformation into myofibroblasts. Bundles of spindle-shaped myofibroblasts accumulated beneath the chorionic epithelium. These cells were strongly positive for alphaSMA and FN, but negative for desmin. They possessed a well developed rough endoplasmic reticulum and bundles of microfilaments anchoring in the cell membrane. Our results suggest that PDGF-B is a "transforming" growth factor with important functions during formation of granulation tissue which are closely comparable to the effects of the PDGF-B-like protein of simian sarcoma virus. PDGF-B also induced vascular alterations in the CAM, which, however, appeared to be a secondary effect. While the intra-chorionic capillaries were lost, an accumulation of small vessels positive for alphaSMA was observed. This indicates a function for PDGF-B during segregation of main vessels from a primary vascular plexus.

The optimum solvent for Nb2Se9 dispersion, which is a new type of one dimensional (1D) material, is investigated. Among several solvents (16 solvents in total), strong dispersion was observed in benzyl alcohol, isopropyl alcohol, isobutyl alcohol, and diacetone alcohol, which have medium dielectric constants in the range of 10 to 30 and surface tension in the range of 25 to 35 mJ m−2. 1D Nb2Se9 chains, whose size is less than 10 nm, are well dispersed and it is possible to disperse mono-chains of 1 nm or less in a specific dispersion region. The 1D unit chain with dangling bond free surface and high volume to area ratio is expected to be used in applications that utilize the surface of the material. Such dispersion is an important first step towards various potential applications and is an indispensable scientific goal for the practical applications of Nb2Se9.