Vascular endothelial growth factor (VEGF) plays a vital role in the development and maintenance of placental function throughout pregnancy and is implicated in complications of pregnancy, affecting the placenta. Pre-eclampsia (PE) is a leading cause of maternal morbidity and mortality, affecting 5-10 % of first pregnancies worldwide. VEGF has been shown to be significantly elevated in the serum of pregnant women before the onset of pre-eclampsia (Hunter et al. 2000). This study looks at two VEGF isoforms: VEGF165, and a recently discovered isoform, VEGF165b, (Bates et al. 2002), which is thought to be anti-angiogenic.
Placental biopsies were obtained post-birth or from Caesarean section from uncomplicated pregnancies (n = 19) and pregnancies complicated by PE (n = 14). PE was determined as in Hunter et al. (2000). Ethical approval was granted by The North Bristol NHS Trust Ethics Committee and written consent obtained from all volunteers.
Total RNA was extracted from 100-200 mg of tissue homogenised in Trizol reagent (Invitrogen Life Technologies), using the method of Chomczynski & Sacchi (1997). 10 µg of mRNA was reverse transcribed using 250U Moloney murine leukaemia virus RT (Abgene) and 0.5 µg Oligo dT primer (Invitrogen Custom Primers) at 37 °C for 1.5 h in 20 µl volume.
cDNA was amplified using primers to specifically detect each isoform and a pan-specific primer that detects all isoforms of VEGF. 1 µg of cDNA was used per 20 µl reaction, with 1.5 mM MgCl2, 1 µM forward and reverse primer, 200 µM dNTPs and 0.5 U Thermoprime plus DNA Polymerase (Abgene). PCR protocols were optimised: VEGF165, 65 °C annealing for 40 cycles producing an amplicon of 200 bp; VEGF165b, 69 °C annealing for 35 cycles producing an amplicon of 220 bp. 1 ng of each isoforms’ cloned cDNA was included as a control.
Data were analysed using Fisher’s exact test. No significant difference (P = 1.00), was found between the two groups of placental samples in the expression of VEGF165 (7/14 PE and 9/19 normal positive); or VEGF165b (6/14 PE and 4/19 normal positive). However, VEGF165 was expressed at a significantly higher frequency than VEGF165b in normal patients. All samples were positive for at least one isoform of VEGF using pan-specific primers. In conclusion, we have shown in this study that VEGF165 and VEGF165b are not seen in increased frequency in placenta affected by pre-eclampsia than in placenta from uncomplicated pregnancy, but that neither isoform is present in up to half of normal placentae.