BioMed Research International / 2014 / Article / Fig 3

Research Article

Angiogenin Expression during Early Human Placental Development; Association with Blood Vessel Formation

Figure 3

Angiogenin expression by trophoblastic cells. (A) Expression of angiogenin transcripts by trophoblasts isolated from second-trimester placenta. RT-PCR detection of a 264 bp fragment of the angiogenin transcript on 2% agarose gel stained with ethidium bromide. Lanes 1, 3, and 5: cytotrophoblasts from 15-, 16-, and 17-week placentas prior to culture (CT0) and 2, 4, and 6: RNA from 72 h cultured cytotrophoblasts, respectively, differentiated in vitro into a syncytiotrophoblast (ST); 7 is a negative control without RNA; 8 is a positive control using human liver RNA. -actin PCR gave a product at 371 bp. (B) Angiogenin release by cultured villous cytotrophoblasts from 14-week placenta. Angiogenin (ANG) was released into the culture medium during in vitro differentiation of cytotrophoblasts into a syncytiotrophoblast (a). Human choriogonadotropin (hCG) was maximally expressed on day 3, indicating a functional syncytium (b). Results are means ± SD of triplicate determinations in a representative experiment ( ; ; ). (C) Angiogenin immunodetection in cultured cytotrophoblasts from first-trimester chorionic villi in vitro. The cells were fixed with paraformaldehyde and permeabilized and then reacted with monoclonal antiangiogenin. The bound antibody was revealed with FITC-conjugated goat anti-mouse IgM (mANG, in green). Nuclei were counterstained with Dapi (in blue). Angiogenin staining increased with cell differentiation: villous cytotrophoblasts at day 1 (a) compared to the cells at day 2 (b); control with nonspecific mouse IgM was negative (c). Angiogenin labelling was heterogeneous: diffuse in single cells (e, arrowhead), dense and more pronounced around nuclei in aggregating cells (b, e, and white arrow), punctuated and associated with granules here in the syncytium (d, yellow arrow). Cells were from 13-week (a, b, and c), 12.5-week (d), and 8.5-week (e) placenta, respectively. Bar, 20 μm.