Histomorphology and vascular lesions in dorsal rat skin used as injection sites for a subcutaneous toxicity study.

Subcutaneous injection of pharmaceutical compounds into the dorsal skin of rats is common in preclinical and nonclinical studies. However, no detailed histologic description of this anatomic location has been published to date. Following the observation of vascular lesions in the dorsum of rats in a thirteen-week toxicity study, a complementary study was performed on untreated Sprague-Dawley rats to evaluate the normal histology of the skin and subcutis, the potential effect of chronic subcutaneous injection on the morphology of the skin and its vasculature, and the spontaneous vascular pathology in the areas used as injection sites in the principal study. This study showed that saline injection did not fundamentally alter the morphology of the injection sites used for the principal study. Skin thickness was greater in males than in females. Although acellular intimal thickening occurred spontaneously in the dorsal skin of untreated males and females, only males had a spontaneous incidence of intimal hyperplasia. No site predilection for intimal lesions was apparent for either sex. Saline injection, or the physical trauma of injection, may induce intimal hyperplasia; males appear more likely to develop the lesion than do females. It is possible that acellular intimal thickening can progress to intimal hyperplasia under appropriate conditions.

Functional and placental expression analysis of the human NRF3 transcription factor.

Members of the Maf protooncogene and cap'n' collar families of basic-leucine zipper transcription factors play important roles in development, differentiation, oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basic-leucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/MAFG heterodimer recognizes nuclear factor-erythroid 2/Maf recognition element-type DNA-binding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We found that NRF3 transcripts are present in placental chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocarcinoma cell lines BeWo and JAR, derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3-specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF-alpha in JAR cells. Our functional studies suggest that human NRF3 is a potent transcriptional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development.

Human cytotrophoblasts promote endothelial survival and vascular remodeling through secretion of Ang2, PlGF, and VEGF-C.

Cytotrophoblasts are specialized epithelial cells of the human placenta that differentiate to acquire tumor-like properties that allow them to invade the uterus. Concurrently, they develop endothelial-like characteristics. This transformation allows cytotrophoblasts to replace the maternal cells that line uterine vessels, thereby diverting maternal blood to the placenta. Previously, we showed that invading cytotrophoblasts secrete VEGF-C and PlGF, factors that regulate their acquisition of an endothelial-like phenotype. Here, we examined the cells' expression of angiopoietin ligands and their Tie receptors. The data show that cytotrophoblasts predominantly expressed Ang2. We also studied the paracrine functions of Ang2 and the VEGFs by culturing uterine microvascular endothelial cells in cytotrophoblast-conditioned medium, which supported their growth. Removal of VEGF-C, PlGF, and/or Ang2 from the medium caused a marked reduction in cell number due to massive apoptosis. We also assayed the angiogenic potential of cytotrophoblast-derived factors in the chick chorioallantoic membrane assay. The results showed that they stimulated angiogenesis to a level comparable to that of basic FGF. These data suggest that invasive human cytotrophoblasts use an unusual repertoire of factors to influence the angiogenic state of maternal blood vessels and that this cross talk plays an important part in the endovascular component of uterine invasion.