Dawn E. Clark

Vascular endothelial growth factor (VEGF) is a potent secreted factor that promotes angiogenesis and maintains the integrity of the endothelium. Levels of VEGF are increased in many tumors and are elevated in women with pre-eclampsia, a serious disease of pregnancy. Here we show by in situ hybridization that the trophoblast contains the mRNA encoding a soluble version of the VEGF receptor known as Flt-1 (sFlt-1: initially described by Kendall and Thomas, PNAS 90:10705-10709). Binding assays and Western blotting of villus-conditioned media confirmed the production of sFlt-1. Serum from pregnant women was found to contain a VEGF-binding protein that was not present in serum from men or nonpregnant women. As determined by heparin affinity, column fractionation, and cross-linking, this protein was identical to sFlt-1. Taken together, these results show that the placenta secretes sFlt-1, which would be expected to be a VEGF antagonist. This is the first report of production of the sFlt-1 receptor in vivo, and it reveals a new mechanism for naturally regulating this potent angiogenic agent. The presence of such an antagonist suggests that regulation of VEGF action is essential to successful pregnancy. This has important implications for the activity of VEGF locally and systemically in other conditions.

Angiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.

The utilization of a deer antler model to study gene expression in tissues undergoing rapid growth has been hampered by an inability to sample the different tissue types. We report here a standardized procedure to identify different tissue types in growing antler tips and demonstrate that it can help in the classification of expressed sequence tags (ESTs). The procedure was developed using observable morphological markers within the unstained tissue at collection, and was validated by histological assessments and virtual Northern blotting. Four red deer antlers were collected at 60 days of growth and the tips (top 5 cm) were then removed. The following observable markers were identified distoproximally: the dermis (4.86 mm), the subdermal bulge (2.90 mm), the discrete columns (6.50 mm), the transition zone (a mixture of discrete and continuous columns) (3.22 mm), and the continuous columns (8.00 mm). The histological examination showed that these markers corresponded to the dermis, reserve mesenchyme, precartilage, transitional tissue from precartilage to cartilage, and cartilage, respectively. The gene expression studies revealed that these morphologically identified layers were functionally distinct tissue types and had distinct gene expression profiles. We believe that precisely defining these tissue types in growing antler tips will greatly facilitate new discoveries in this exciting field.

Annual antler renewal presents the only case of epimorphic regeneration (de novo formation of a lost appendage distal to the level of amputation) in mammals. Epimorphic regeneration is also referred to as a blastema-based process, as blastema formation at an initial stage is the prerequisite for this type of regeneration. Therefore, antler regeneration has been claimed to take place through initial blastema formation. However, this claim has never been confirmed experimentally. The present study set out to describe systematically the progression of antler regeneration in order to make a direct histological comparison with blastema formation. The results showed that wound healing over a pedicle stump was achieved by ingrowth of full-thickness pedicle skin and resulted in formation of a scar. The growth centers for the antler main beam and brow tine were formed independently at the posterior and anterior corners of the pedicle stump, respectively. The hyperplastic perichondrium surmounting each growth center was directly formed in situ by a single type of tissue: the thickening distal pedicle periosteum, which is the derivative of initial antlerogenic periosteum. Therefore, the cells residing in the pedicle periosteum can be called antler stem cells. Antler stem cells formed each growth center by initially forming bone through intramembranous ossification, then osseocartilage through transitional ossification, and finally cartilage through endochondral ossification. There was an overlap between the establishment of antler growth centers and the completion of wound healing over the pedicle stump. Overall, our results demonstrate that antler regeneration is achieved through general wound healing- and stem cell-based process, rather than through initial blastema formation. Pedicle periosteal cells directly give rise to antlers. Histogenesis of antler regeneration may recapitulate the process of initial antler generation.