Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms

Abstract

Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor -1 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined glucose transporter expression and localization in both a mouse model of IUGR and a model of human trophoblast, the BeWo Choriocarcinoma cell line.

Methods: At gestational day 18, animals were divided into four groups; sham-operated controls, uterine artery branch ligation (UABL), UABL+Ad-hIGF-1 (10(8) PFU), UABL+Ad-LacZ (10(8) PFU). At gestational day 20, pups and placentas were harvested by C-section. For human studies, BeWo choriocarcinoma cells were grown in F12 complete medium +10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 hours. MOIs of 10∶1 and 100∶1 were utilized. The RNA, protein expression and localization of glucose transporters GLUT1, 3, 8, and 9 were analyzed by RT-PCR, Western blot and immunohistochemistry.

Results: In both the mouse placenta and BeWo, GLUT1 regulation was linked to altered protein localization. GLUT3, localized to the mouse fetal endothelial cells, was reduced in placental insufficiency but maintained with Ad-I GF-1 treatment. Interestingly, GLUT8 expression was reduced in the UABL placenta but up-regulated following Ad-IGF-1 in both mouse and human systems. GLUT9 expression in the mouse was increased by Ad-IGF-1 but this was not reflected in the BeWo, where Ad-IGF-1 caused moderate membrane relocalization.

Conclusion: Enhanced GLUT isoform transporter expression and relocalization to the membrane may be an important mechanism in Ad-hIGF-1mediated correction of placental insufficiency.

Figure 1. Summary of GLUT1, 3, 8 and 9 mRNA expression in control, UABL, Ad-hIGF-1 and Ad-LacZ treated mouse placentas.

ANOVA, Post hoc Tukeys test * p<0.05, n>3 for each treatment

Figure 2. Representative micrographs of GLUT1 (A,B,C), GLUT3 (D,E,F), GLUT8 (G,H,I), GLUT9a (J,K,L) and GLUT9b (M,N,O) in mouse placental labyrinth in the 3 treatment groups, Sham, UABL and Ad-hIGF-1 respectively.

100× magnification. ST identifies syncytiotrophoblast, * identifies a fetal villous vessel, # identifies a maternal blood sinus. Micrographs are representative of 3 biological replicates for each treatment group.

Figure 3. Localization of GLUT8 in human placenta.

A) Rabbit IgG control demonstrating no staining in human placenta. B) GLUT8 is expressed in the syncytiotrophoblast (closed arrows) and the villous vessel endothelium (black arrowheads) in normal, term, human placenta.

Figure 4. GLUT8 mRNA expression is significantly increased following exposure of BeWo cells to Ad-IGF-1 at an MOI of 100∶1 for 48 hours.

ANOVA, Tukeys posthoc test ***p<0.005, n = 6 replicates

Figure 5. GLUT expression levels in BeWo cells.

A:Representative Western blots of GLUT1, 3, and 8 in BeWo cells treated with control media (C), Ad-hIGF-1 MOI 10∶1 (10), Ad-hIGF-1 MOI 100∶1 (100) for 48 hours. B: Summary of GLUT1, 3, 8 protein expression. ANOVA, Tukeys posthoc test, *p<0.05, ** p<0.01, n>4 replicates for each treatment.

Figure 6. GLUT1 localization is shown in control cells (A) and Ad-hIGF-1 (B) treated BeWo cells1.

GLUT3 is localized to both the perinuclear region and the cell membrane in both control (C) and Ad-hIGF-1 (D) treated BeWo.GLUT8 protein expression is minimal in control BeWo (E) but increased following exposure to Ad-hIGF-1 (F). Under control conditions GLUT9a remains perinuclear (G) but following exposure to Ad-hIGF-1 -localizes to the cell membrane (H). GLUT9b is distributed throughout the cell membrane in control (I) and Ad-hIGF-1 (J) treated BeWo cells. Images are representative of at least 3 different passages of BeWo cells.