Increased vascular and uteroplacental matrix metalloproteinase-1 and -7 levels and collagen type I deposition in hypertension in pregnancy: role of TNF-α

Abstract

Preeclampsia is a pregnancy-related disorder manifested as maternal hypertension in pregnancy (HTN-Preg) and fetal growth restriction. Placental ischemia could be an initiating event that leads to abnormal vascular and uteroplacental remodeling in HTN-Preg; however, the molecular targets and intermediary mechanisms involved are unclear. We tested the hypothesis that placental ischemia could target vascular and uteroplacental matrix metalloproteinases (MMPs) through an inflammatory cytokine-mediated mechanism. MMP levels and distribution were measured in the aorta, uterus, and placenta of normal pregnant (Preg) rats and pregnant rats with reduced uterine perfusion pressure (RUPP). Maternal blood pressure was higher and the litter size and pup weight were lower in RUPP compared with Preg rats. Gelatin zymography showed prominent uterine MMP-2 and MMP-9 activity that was dependent on the amount of loaded protein. At saturating protein loading, both gelatin and casein zymography revealed two additional bands corresponding to MMP-1 and MMP-7 that were greater in the aorta, uterus, and placenta of RUPP compared with Preg rats. Western blots and immunohistochemistry confirmed increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of RUPP versus Preg rats. The levels of MMP-1 and MMP-7 substrate collagen type I were greater in tissues of RUPP compared with Preg rats. In organ culture, TNF-α increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of Preg rats, and a TNF-α antagonist prevented the increases in MMPs in tissues of RUPP rats. Thus, placental ischemia, possibly through TNF-α, increases vascular and uteroplacental MMP-1 and MMP-7, which, in turn, alter collagen deposition and cause inadequate tissue remodeling in HTN-Preg. Cytokine antagonists may reverse the increase in MMP-1 and MMP-7 expression/activity and, in turn, restore proper vascular and uteroplacental remodeling in HTN-Preg and preeclampsia.NEW & NOTEWORTHY The molecular mechanisms of preeclampsia are unclear, making it difficult to predict, prevent, or manage the pregnancy-associated disorder. This study showed that placental ischemia, possibly through the release of TNF-α, causes increases in the levels of matrix metalloproteinase (MMP)-1 and MMP-7, which could alter collagen deposition and cause inadequate uteroplacental and vascular remodeling in hypertension in pregnancy. The data suggest that targeting MMP-1 and MMP-7 and their upstream modulators, such as TNF-α, could provide a new approach in the management of hypertension in pregnancy and preeclampsia.

Keywords: aorta; placenta; preeclampsia; remodeling; tumor necrosis factor-α; uterus.

Fig. 1.

Concentration-dependent gelatinase activity in the aorta, uterus, and placenta of pregnant rats with reduced uterine perfusion pressure (RUPP). Aortic (A), uterine (B), and placental tissue strips (C) from RUPP rats were homogenized and prepared for gelatin zymography analysis using different amounts of loaded protein (0.1–10 µg). The proteolytic bands corresponding to pro-matrix metalloproteinase (MMP)-2 and MMP-2 as well as MMP-9 were not analyzed as they were the topic of a previous report (41). The densitometry values of the proteolytic bands corresponding to MMP-1 and MMP-7 are presented as pixel intensity × mm². Bar graphs represent means ± SE; n = 4/group. *MMP-1 or MMP-7 activity at a specific amount of loaded protein was significantly different (P < 0.05) from the preceding protein amount.

Fig. 2.

Aortic, uterine, and placental MMP-1 and MMP-7 gelatinase activity in normal pregnant (Preg) and RUPP rats. Equal protein amount (5 μg) of tissue homogenates from the aorta (A), uterus (B), and placenta (C) of Preg and RUPP rats were prepared for gelatin zymography analysis. At 5 µg loaded protein amount, the proteolytic bands corresponding to pro-MMP-2 and MMP-2 in the aorta and uterus were saturated, and no differences could be detected between the groups. The proteolytic bands corresponding to pro-MMP-2 and MMP-2 in the placenta appeared to be reduced in RUPP versus Preg rats, confirming our previous report (41). The densitometry values of the proteolytic bands corresponding to MMP-1 and MMP-7 are presented as pixel intensity × mm² and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 6/group. *Measurements in RUPP rats were significantly different (P < 0.05) from the corresponding measurements in Preg rats.

Fig. 3.

Concentration-dependent caseinase activity in the uterus of Preg rats. Uterine tissue strips from Preg rats were homogenized and prepared for casein zymography analysis using different concentrations of casein in gel (0.2%, 0.4%, 0.6%, 0.8%, 1%, and 2%) as well as different amounts of loaded protein (0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 µg). The densitometry values of the proteolytic bands corresponding to MMP-1 and MMP-7 are presented as pixel intensity × mm². Data points represent means ± SE; n = 4/group. *MMP-1 or MMP-7 activity at a specific amount of loaded protein was significantly different (P < 0.05) from the activity at the preceding protein amount.

Fig. 4.

Aortic, uterine, and placental MMP-1 and MMP-7 caseinase activity in Preg and RUPP rats. Equal protein amount (2 μg) of tissue homogenates from the aorta (A), uterus (B), and placenta (C) of Preg and RUPP rats were prepared and run in duplicates for casein zymography analysis. The densitometry values of the proteolytic bands corresponding to MMP-1 and MMP-7 are presented as pixel intensity × mm² and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 4/group. *Measurements in RUPP rats were significantly different (P < 0.05) from the corresponding measurements in Preg rats.

Fig. 5.

Protein amount of aortic, uterine, and placental MMP-1 and MMP-7 in Preg and RUPP rats. Tissue homogenates of the aorta (A), uterus (B), and placenta (C) of Preg and RUPP rats were prepared for Western blot analysis using antibodies to MMP-1 (1:1,000) and MMP-7 (1:1,000). Immunoreactive bands corresponding to MMP-1 and MMP-7 were analyzed by optical densitometry and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 4/group. *Measurements in RUPP rats were significantly different (P < 0.05) from corresponding measurements in Preg rats.

Fig. 6.

Distribution of MMP-1 and MMP-7 in the aorta of Preg and RUPP rats. Cryosections (6 μm) of the aorta of Preg (A) and RUPP rats (B) were prepared for hematoxylin and eosin (H&E) staining or immunohistochemical staining using MMP-1 or MMP-7 antibodies (1:100). For MMP immunostaining, the total number of pixels in the tissue section wall was first defined and the number of brown spots (pixels) was then counted and presented as a percentage of the total wall area (C and D). The number of pixels in the specific vascular layer (intima, media, and adventitia) was also defined and transformed into the area in mm² using a calibration bar. The number of brown spots (pixels) representing MMP-1 and MMP-7 in each vascular layer was then counted and presented as number of pixels per mm² (E and F). Total magnification: ×400. Bar graphs represent means ± SE; n = 4−6/group. *Measurements in RUPP rats were significantly different (P < 0.05) from the corresponding measurements in Preg rats.

Fig. 7.

Distribution of MMP-1 and MMP-7 in the uterus of Preg and RUPP rats. Cryosections (6 μm) of the uterus of Preg (A) and RUPP rats (B) were prepared for H&E staining or immunohistochemical staining using MMP-1 or MMP-7 antibodies (1:100). Because the rat uterus is large, 10−16 picture frames of sequential parts of the uterine tissue section were acquired using a ×4 objective and inserted as individual raw images in Powerpoint. The image frames were aligned and then grouped and saved as a composite JPG image of the whole reconstituted uterine tissue section for image analysis using ImageJ software. For MMP immunostaining, the total number of pixels in the tissue section wall was first defined and the number of brown spots (pixels) was then counted and presented as a percentage of the total wall area (C and D). Total magnification: ×40. Bar graphs represent means ± SE; n = 4−6/group. *Measurements in RUPP rats were significantly different (P < 0.05) from the corresponding measurements in Preg rats.

Fig. 8.

Distribution of MMP-1 and MMP-7 in the placenta of Preg and RUPP rats. Cryosections (6 μm) of the placenta of Preg (A) and RUPP rats (B) were prepared for H&E or immunohistochemical staining using MMP-1 or MMP-7 antibodies (1:100). Because the rat placenta is large, 10−16 picture frames of sequential parts of the placental tissue section were acquired using a ×4 objective and the composite image of the whole placental tissue section was reconstituted and analyzed using ImageJ software. For MMP immunostaining, the total number of pixels in the tissue section image was first defined and the number of brown spots (pixels) was then counted and presented as a percentage of the total area (C and D). Total magnification: ×40. Bar graphs represent means ± SE; n = 4−6/group. *Measurements in RUPP rats were significantly different (P < 0.05) from the corresponding measurements in Preg rats.

Fig. 9.

Protein levels of aortic, uterine, and placental collagen type I in Preg and RUPP rats. Tissue homogenates of the aorta (A), uterus (B), and placenta (C) of Preg and RUPP rats were prepared and run in duplicate for Western blot analysis using antibody to collagen type I (1:1,000). Immunoreactive bands corresponding to collagen were analyzed by optical densitometry and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 4/group. *Measurements in RUPP rats were significantly different (P < 0.05) from the corresponding measurements in Preg rats.

Fig. 10.

Effect of the cytokine TNF-α and TNF-α antagonist on gelatinase activity of aortic, uterine, and placental MMP-1 and MMP-7 in Preg and RUPP rats. Aortic (A), uterine (B), and placental strips (C) of Preg rats were treated with TNF-α (0.1 μg/ml) with or without TNF-α antagonist (0.1 μg/ml), and tissues of RUPP rats were treated with TNF-α antagonist for 48 h in organ culture. Tissue homogenates were prepared and loaded at 5 µg protein amount for gelatin zymography analysis. At 5 µg amount of loaded protein, the proteolytic bands corresponding to pro-MMP-2 and MMP-2 were saturated, and no differences could be detected between the treatment groups. The densitometry values of the proteolytic bands corresponding to MMP-1 and MMP-7 are presented as pixel intensity × mm² and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 6/group. *Significantly different (P < 0.05) from corresponding measurements in control nontreated tissues of Preg rats.

Fig. 11.

Effect of the cytokine TNF-α and TNF-α antagonist on protein amount of aortic, uterine, and placental MMP-1 and MMP-7 in Preg and RUPP rats. Tissue homogenates of the aorta (A), uterus (B), and placenta (C) of Preg rats were treated with TNF-α (0.1 μg/ml) with or without TNF-α antagonist (0.1 μg/ml), and tissues of RUPP rats were treated with TNF-α antagonist for 48 h in organ culture. Tissues were homogenized and prepared for Western blot analysis using antibody to MMP-1 (1:1,000) or MMP-7 (1:1,000). Immunoreactive bands corresponding to MMP-1 and MMP-7 were analyzed by optical densitometry and normalized to β-actin to correct for loading. Bar graphs represent means ± SE; n = 6/group. *Significantly different (P < 0.05) from corresponding measurements in control nontreated tissues of Preg rats.