Spatio-temporal expression of MMP-2, MMP-9 and tissue kallikrein in uteroplacental units of the pregnant guinea-pig (Cavia porcellus)

Abstract

Background: In humans trophoblast invasion and vascular remodeling are critical to determine the fate of pregnancy. Since guinea-pigs share with women an extensive migration of the trophoblasts through the decidua and uterine arteries, and a haemomonochorial placenta, this species was used to evaluate the spatio-temporal expression of three enzymes that have been associated to trophoblast invasion, MMP-2, MMP-9 and tissue kallikrein (K1).

Methods: Uteroplacental units were collected from early to term pregnancy. MMP-2, MMP-9 and K1 were analysed by immunohistochemistry and Western blot. The activities of MMP-2 and MMP-9 were assessed by gelatin zymography.

Results: Immunoreactive MMP-2, MMP-9 and K1 were detected in the subplacenta, interlobar and labyrinthine placenta, syncytial sprouts and syncytial streamers throughout pregnancy. In late pregnancy, perivascular or intramural trophoblasts expressed the three enzymes. The intensity of the signal in syncytial streamers was increased in mid and late pregnancy for MMP-2, decreased in late pregnancy for MMP-9, and remained stable for K1. Western blots of placental homogenates at days 20, 40 and 60 of pregnancy identified bands with the molecular weights of MMP-2, MMP-9 and K1. MMP-2 expression remained constant throughout gestation. In contrast, MMP-9 and K1 attained their highest expression during midgestation. Placental homogenates of 20, 40 and 60 days yielded bands of gelatinase activity that were compatible with MMP-2 and MMP-9 activities. ProMMP-2 and MMP-9 activities did not vary along pregnancy, while MMP-2 and MMP-9 increased at 40 and 40-60 days respectively.

Conclusion: The spatio-temporal expression of MMPs and K1 supports a relevant role of these proteins in trophoblast invasion, vascular remodeling and placental angiogenesis, and suggests a functional association between K1 and MMP-9 activation.

Figure 1

In subsequent sections of a uteroplacental unit obtained in day 15 of pregnancy, the multilayered subplacenta (SP), and the initial sprouts that give origin to the placenta, expressed K1, MMP-2 and MMP-9. Syncytiotrophoblasts composing the interlobium and the labyrinth expressed K1, MMP-2 and MMP-9 in sections obtained in days 20, 40 and 60 of pregnancy. Bar = 50 μm.

Figure 2

Western blot analysis of guinea-pig placental homogenates for MMP-2. A, Representative blot at days 20, 40 and 60 of pregnancy. Purified human proMMP-2 (hProMMP-2) yielded two bands with approximate molecular weights of 72 and 62 kDa. B, Mean ratio of active/total MMP-2 in three placental homogenates for each period showed no significant differences. DSCF posthoc comparisons after Kruskal-Wallis test.

Figure 3

Western blot analysis of guinea-pig placental homogenates for MMP-9. A, Representative blot at days 20, 40 and 60 of pregnancy. Purified human proMMP-9 (hProMMP-9) yielded two bands with approximate molecular weights of 92 and 68 kDa. B, Mean ratio of active/total MMP-9 in five placental homogenates for each period. **P < 0.01 for 20 vs. 40 days; ***P < 0.001 for 40 vs. 60 days. DSCF posthoc comparisons after Kruskal-Wallis test.

Figure 4

Western blot analysis of guinea-pig placental homogenates for tissue kallikrein (K1). A, Representative blot at days 20, 40 and 60 of pregnancy. Purified rat K1 (rK1) was detected as a broad band with an approximate molecular weight of 31 kDa. B, Densitometric analysis of K1 expression in five placental homogenates for each period. Data is expressed with respect to the average densitometry at day 20. *, P < 0.05 for 60 vs. 40 days; ***, P < 0.001 for 40 vs. 20 days. DSCF posthoc comparisons after Kruskal-Wallis test.

Figure 5

Gelatin zymography of placental homogenates. A, Representative zymogram at days 20, 40 and 60 of pregnancy. Purified human proMMP-2 (hproMMP-2) and proMMP-9 (hproMMP-9) were used as standards. B-C, Densitometric analysis of zymograms from three placental homogenates at days 20, 40 and 60 for Pro/MMP-2 (B) and Pro/MMP-9 (C). Data is expressed with respect to the average densitometry at day 20. *, P < 0.05 for 40 vs. 20 and 60 days in (B); *, P < 0.05 for 40 and 60 vs. 20 days in (C). DSCF posthoc comparisons after Kruskal-Wallis test.

Figure 6

Trophoblasts characterized by cytokeratin (CK) in the subplacenta (SP) and in the decidual syncytial streamers (ST) expressed MMP-2, MMP-9 and K1 in sequential sections of days 15, 20 and 40 of pregnancy. Bar = 50 μm

Figure 7

Spiral arteries observed in sequential sections of myometrium obtained in days 40 and 60 of pregnancy, expressed MMP-2, MMP-9 and K1 in trophoblasts surrounding the vessel, characterized by cytokeratin (CK), and in the swollen endothelial cells, characterized by von Willebrand Factor (vWF). The remaining vascular smooth muscle in day 40 was positive for muscle actin (MA), and has been replaced in day 60. Bar = 50 μm

Figure 8

Mesometrial arteries obtained in day 40 of pregnancy showed in sequential sections an intact smooth muscle layer characterized by muscle actin (MA), and no cytokeratin positive cells. In day 60, intramural trophoblasts, characterized by cytokeratin (CK), expressed MMP-2, MMP-9 and K1, while endothelial cells, identified by von Willebrand Factor (vWF), were swollen, and expressed MMP-9 and K1. The remaining vascular smooth muscle, positive for muscle actin (MA), was disrupted. Bar = 50 μm

Figure 9

Control sections at different days of pregnancy, incubated in absence of the first antibody for subplacenta (A), interlobium and labyrinth (B-G), subplacenta and syncitial streamers (H), spiral arteries (I,J) and mesometrial artery (K). Bar = 50 μm