Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro

Abstract

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1β, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption.

Figure 1

Effect of Increasing Thrombin Dose upon FM Mechanical Properties: Thrombin (0–100 u/ml) induced concentration-dependent decreases in FM rupture strength (upper panel), stiffness (middle panel) and work to rupture (bottom panel). All incubations were for 48 h. The data shown represent triplicate FM cultures repeated three times using three different placentas. (Data are presented mean ± SD, * p < 0.001 vs. no thrombin control)

Figure 2

Thrombin Induces MMP9 and MMP3 in Amnion: Thrombin (0–100 u/ml for 48 h) induced concentration-dependent increases in both the pro-MMP9 (92 kD) and the active MMP9 (78 kD) protein (Panel A); and in both the pro-MMP3 (57 kD) and the active MMP3 (45 kD) protein (Panel B); in the amnion component of treated FM explants. The Western blot shown is representative of three replicate experiments with three different placentas. To confirm the significance of qualitative results, triplicate blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods. (Data are presented mean ± SD, * p < 0.01 vs. no thrombin control)

Figure 2

Thrombin Induces MMP9 and MMP3 in Amnion: Thrombin (0–100 u/ml for 48 h) induced concentration-dependent increases in both the pro-MMP9 (92 kD) and the active MMP9 (78 kD) protein (Panel A); and in both the pro-MMP3 (57 kD) and the active MMP3 (45 kD) protein (Panel B); in the amnion component of treated FM explants. The Western blot shown is representative of three replicate experiments with three different placentas. To confirm the significance of qualitative results, triplicate blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods. (Data are presented mean ± SD, * p < 0.01 vs. no thrombin control)

Figure 3

Thrombin effect upon MMP9 and MMP3 in Choriodecidua: Thrombin (0–100 u/ml for 48 h) failed to show concentration-dependent increases in pro-MMP9 (92 kD) in the choriodecidua component of treated FM explants. The active MMP9 (78 kD) form was not detected (Panel A). Thrombin (0–100 u/ml for 48 h) failed to show concentration-dependent increases in either the pro-MMP3 (57 kD) or the active MMP3 (45 kD) protein in the choriodecidual component of treated FM explants (Panel B). The Western blot shown is representative of three replicate experiments with three different placentas. To confirm the significance of qualitative results, triplicate blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods. (Data are presented mean ± SD, no significant differences were detected.)

Figure 3

Thrombin effect upon MMP9 and MMP3 in Choriodecidua: Thrombin (0–100 u/ml for 48 h) failed to show concentration-dependent increases in pro-MMP9 (92 kD) in the choriodecidua component of treated FM explants. The active MMP9 (78 kD) form was not detected (Panel A). Thrombin (0–100 u/ml for 48 h) failed to show concentration-dependent increases in either the pro-MMP3 (57 kD) or the active MMP3 (45 kD) protein in the choriodecidual component of treated FM explants (Panel B). The Western blot shown is representative of three replicate experiments with three different placentas. To confirm the significance of qualitative results, triplicate blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods. (Data are presented mean ± SD, no significant differences were detected.)

Figure 4

Lipoic Acid (LA) inhibits Thrombin induced-weakening: FM fragments were pre-incubated with or without LA (0.25 mM) for 6h, then incubated with or without the addition of Thrombin (10 u/ml) for 48h. Four groups are displayed: Control, LA pre-treatment alone, Thrombin alone, LA treatment + Thrombin. Rupture Strength (upper panel), Stiffness (middle panel) and Work to Rupture (lower panel) were determined as outlined in Methods. Data points represent pooled results of three experiments using three FM fragments per treatment group in each experiment. (Data are presented mean ± SD, * p < 0.001 vs. all other groups)

Figure 5

Lipoic Acid (LA) inhibits Thrombin induced increases in MMP9 in the amnion component of cultured FM. Intact FM fragments were pre-incubated with or without LA (0.25 mM) for 6h, then incubated with or without the addition of thrombin (10 u/ml) for 48h. After the incubations, the amnion was then separated from the choriodecidua and tested. Four groups are displayed: Control, LA pre-treatment, thrombin, LA treatment + thrombin. The Western blot shown is representative of three replicate experiments using three different placentas . Both the pro-MMP9 (92 kD) and the active MMP9 (78 kD) protein in the amnion component of treated FM explants are shown. To confirm the significance of qualitative results, blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods (Data are presented mean ± SD, * p < 0.01 vs. all other groups).

Figure 6

Lipoic Acid (LA) inhibits Thrombin induced increases in MMP3 in the amnion component of cultured FM. Intact FM fragments were pre-incubated with or without LA (0.25 mM) for 6h, then incubated with or without the addition of Thrombin (10 u/ml) for 48h. After the incubations, the amnion was then peeled from the choriodecidua and tested. Four groups are displayed: Control, LA pre-treatment, Thrombin, LA treatment + Thrombin. The Western blot shown is representative of three replicate experiments with three different placentas (N=9). Both the pro-MMP3 (57 kD) and the active MMP3 (45 kD) protein in the amnion component of treated FM explants are shown. To confirm the significance of qualitative results, blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods (Data are presented mean ± SD, * p < 0.01 from all groups, + p < 0.05 from control).