Purpose: Pericytes are known to provide a survival advantage for endothelial cells.We hypothesize that strategies aimed at dual targeting of tumor-associated endothelial cells and pericytes will be highly efficacious. Experimental Design: Paclitaxel-sensitive (HeyA8 and SKOV3ip1) or paclitaxel-resistant (HeyA8-MDR) orthotopic tumors in mice were examined for therapeutic efficacy by targeting the endothelial cells (using a vascular endothelial growth factor receptor inhibitor, AEE788) and pericytes (using STI571) alone or in combination. Additional therapy and survival studies in combination with paclitaxel were also done. Following therapy, tumors were examined for endothelial cell apoptosis, pericyte coverage, microvessel density, and proliferation. Results: AEE788 inhibited tumor growth by 45% and 59% in the HeyA8 and SKOV3ip1models, respectively, whereas STI571 alone was not effective. AEE788 plus STI571 resulted in 69% to 84% inhibition of tumor growth in both models. Moreover, combination of these agents with paclitaxel was even more effective, resulting in up to 98% inhibition of tumor growth. The triple combination was even effective in the HeyA8-MDR model. Remarkably, this triple combination also resulted in improved survival compared with all other groups (P < 0.001) and caused regression of formed tumors. Pericyte coverage was significantly decreased in the STI571 treatment groups, and microvessel density was significantly reduced in the AEE788 treatment groups. AEE788 induced endothelial cell apoptosis, which was further enhanced by the addition of STI571. Conclusions: Strategies targeting both endothelial cells and pericytes are highly effective for in vivo treatment of ovarian carcinoma. This antiangiogenic effect may be partially due to decreased pericyte coverage, thus increasing the sensitivity of tumor vasculature to therapy. These encouraging data support the development of clinical trials based on this strategy.

The present study was undertaken to examine the development of the trochlear nucleus in quail and to compare the mature trochlear nucleus, nerve, and their sole target of innervation, the superior oblique muscle, in quail, chick, and duck. Study of the trochlear nucleus in quail from embryonic day 5 through hatching shows a maximum of 1,248 neurons on embryonic day 10 followed by spontaneous degeneration of 40% of the neurons between days 10 and 16. Previous studies have shown that although the initial and final number of neurons is different in chick and duck, the magnitude of trochlear cell loss in both species is about 40%. This study shows the average number of neurons in the nucleus of quail, chick, and duck, 2 weeks post-hatching, to be 658, 743 and 1,459, respectively. Fiber counts in the trochlear nerve from electron micrograph montages at the same period indicated a ratio of about 1:1 between neurons and axons. While a majority of the fibers in these nerves are myelinated, an average of 3-6% of the fibers are unmyelinated. The nucleus in the quail not only contains the smallest number of neurons but it also innervates the smallest muscle in terms of total number of muscle cells and endplates. However, the opposite relationship does not hold true. The nucleus in duck contains the largest number of neurons, yet the largest number of muscle cells and endplates were found in the chick. The ratios between the neurons and muscle cells as well as between neurons and endplates are about the same in quail and duck. These ratios are much higher in the chick, reflecting the relatively small neuron pool destined for a relatively large target. In spite of variations in the number of neurons, muscle fibers, and endplates the average number of endplates per muscle fiber is relatively constant among the three species.

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The present investigation was undertaken to examine the role of peripheral competition in survival of motor neurons during development. A loss of approximately half of the trochlear motor neurons in duck and quail occurs during the course of normal embryogenesis. The number of motor neurons in the nucleus of quail prior to the onset of cell death is identical to the final number of survivors in the nucleus of duck embryos (about 1,300 neurons). In the present study competition at the peripheral target was decreased by reducing the number of trochlear motor neurons initially projecting to their target muscle. This was accomplished by substituting the midbrain of duck embryos with the same neural tissue from quail embryos. Midbrain transplantation was performed before motor axon outgrowth and normal cell death begin. The development of the motor neurons and their sole target of innervation, the superior oblique muscle, was examined by using a variety of techniques. The source of the grafted motor neurons and of a reduction in the size of the motor neuron pool was confirmed from histological sections and cell counts. The grafted motor neurons projected their axons into the appropriate peripheral target, which was determined by the use of HRP tracing technique. Counts of muscle fibers, motor endplates, and acetylcholine receptors and measurement of total muscle protein indicated that the size of the superior oblique muscle in the chimera embryos was similar to that of the normal duck but significantly larger than the muscle in quail embryos. Electrophysiological observations indicated that the grafted trochlear motor neurons made functional connections with the superior oblique muscle. Counts of the trochlear motor neurons after the period of cell death indicated an average of 1,310 neurons in the nucleus of duck, 772 in quail, and 690 in the chimera embryos. The number of motor neurons in the chimera embryos is not significantly different from that in the normal quail. In other words, in spite of reduced peripheral competition trochlear motor neuron death of normal magnitude occurred. Lack of increased cell survival in our study suggests that trochlear motor neurons do not compete for survival at the peripheral target.

About half of the trochlear motor neurons in duck and quail embryos die during normal development. In a previous study the role of target muscle in controlling the number of surviving motor neurons was investigated by reducing the number of neurons innervating the muscle. This was accomplished by removing the midbrain of the duck embryo and grafting in its place the midbrain of the quail embryo before motor neuron death begins. It was observed that the number of surviving trochlear motor neurons in the quail-duck chimera embryos was not significantly different from that of the normal quail. The present investigation was undertaken to determine whether trochlear motor neurons in the chimera embryos received afferent synapses. Brains of duck, quail and chimera embryos on days 16 and 20 were processed for electron microscopical observations. Synapses formed on motor neurons of the chimera embryos. Surprisingly, synapses on motor neurons of quail differed from those of duck, both qualitatively and quantitatively. Synapses on the motor neurons of the chimera embryos developed in a fashion similar to that for the duck motor neurons. Our failure to rescue trochlear motor neurons in the chimera embryos suggests that the developing motor neurons may respond to a larger target muscle only if they received a normal complement of afferent synaptic input.

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