Reduced NO signaling during pregnancy attenuates outward uterine artery remodeling by altering MMP expression and collagen and elastin deposition

Abstract

Recent findings indicate that endothelial nitric oxide (NO) plays a key role in uterine artery outward circumferential remodeling during pregnancy. Although the underlying mechanisms are not known, they likely involve matrix metalloproteinases (MMPs). The goal of this study was to examine the linkage among NO inhibition, expansive remodeling, and MMP expression within the uterine vascular wall. Adult female rats were treated with N(G)-nitro-L-arginine methyl ester [L-NAME (LPLN)] beginning on day 10 of pregnancy and until death at day 20 and compared with age-matched controls [late pregnant (LP)]. Mean arterial pressure of LPLN rats was significantly higher than controls. LPLN fetal and placental weights were significantly reduced compared with controls. Main uterine arteries (mUA) were collected to determine dimensional properties (lumen area and wall thickness), collagen and elastin content, and levels of endothelial nitric oxide synthase (eNOS) and MMP expression. Circumferential remodeling was attenuated, as evidenced by significantly smaller lumen diameters. eNOS RNA and protein were significantly (>90%) decreased in the LPLN mUA compared with LP. Collagen and elastin contents were significantly increased in LPLN rats by ∼10 and 25%, respectively, compared with LP (P < 0.05). Both MMP-2 and tissue inhibitors of metalloproteinase-2 as assessed by immunofluorescence were lower in the endothelium (reduction of 60%) and adventitia (reduction of 50%) of LPLN compared with LP mUA. Membrane bound MMP-1 (MT1-MMP) as assessed by immunoblot was significantly decreased in LPLN. These data suggest a novel contribution of MMPs to gestational uterine vascular remodeling and substantiate the linkage between NO signaling and gestational remodeling of the uterine circulation via altered MMP, TIMP-2, and MT1-MMP expression and activity.

Fig. 1.

Endothelial nitric oxide synthase (eNOS) RNA and protein expression is decreased by ∼90% in the late pregnant (LP) N^G-nitro-l-arginine methyl ester (l-NAME)-treated (LPLN) main uterine artery (mUA). A: LP and LPLN mUA were dissected, placed immediately in Trizol reagent for subsequent RNA extraction, and quantitative (q)PCR analysis as described in materials and methods. Hprt1 and Ywhaz were used as control genes for the qPCR. eNOS RNA expression was significantly decreased in the LPLN mUA compared with LP. B: LP and LPLN mUA were dissected and placed in modified RIPA buffer for protein analysis as described in materials and methods. Twenty-five micrograms of protein were loaded on the gel and GAPDH was used as a loading control. Top: representative immunoblot. Bottom: densitometry was used to quantify the immunoblots probed for eNOS and normalized by GAPDH expression. eNOS is expressed relative to GADPH. eNOS is significantly decreased in LPLN-treated mUA. *P < 0.05 by Student's t-test; n = 3 animals per group.

Fig. 2.

Lumen area is decreased and extracellular matrix components are increased in LPLN-treated mUA. A: LP and LPLN arteries were dissected, arterial lumens flushed of blood, fixed, and stained using elastic Van Gieson and Masson's trichrome stain as described in materials and methods. Collagen is stained blue, and elastin is stained dark purple. B: collagen content was assessed using the color threshold capability within MetaMorph set to distinguish between collagen and elastin staining. Collagen staining is significantly increased in LPLN mUA compared with LP (n = 7, both groups). C: elastin staining was determined as for collagen; however, the color threshold was set to only include dark purple stained structures within the section. Elastin staining is significantly increased in LPLN mUA (n = 5, both groups). *P < 0.05 by Student's t-test.

Fig. 3.

l-NAME treatment of LP rats decreases mRNA expression of tissue inhibitors of metalloproteinase-2 (TIMP-2) and membrane bound matrix metalloproteinase-1 (MT1-MMP) but not matrix metalloproteinase-2 (MMP-2) within the mUA. LP and LPLN mUA were dissected, placed immediately in Trizol reagent for subsequent RNA extraction and qPCR analysis as described in materials and methods. Hprt1 and Ywhaz were used as endogenous controls for the qPCR. A: although somewhat reduced, LPLN MMP-2 mRNA is not significantly different from LP (P = 0.17). B: l-NAME significantly decreased TIMP-2 mRNA expression in mUA, compared with LP (P = 0.05). C: MT1-MMP trended be lower in LPLN mUA, although this difference did not achieve statistical significance (P = 0.06). *P < 0.05 by Student's t-test.

Fig. 4.

Endothelial and adventitial MMP-2 protein expression is decreased in mUA in LPLN-treated rats. A: mUAs were dissected from LP and LPLN-treated animals, fixed, paraffin-embedded, cross-sectioned, and analyzed using immunofluorescence to detect both pro and active forms of MMP-2 (red). DAPI was used to stain nuclei (blue). Images were taken at ×25 and ×100 as described in materials and methods. Bar = 50 μm. DAPI images represent the secondary antibody control images where there was no incubation with primary antibody. B: MMP-2 protein immunofluorescence was quantified using the integrated intensity function on MetaMorph software and normalizing for area measured. Relative integrated intensity is expressed as a ratio to LP control per area (μm²). LPLN arteries had significantly decreased MMP-2 expression in the endothelial and adventitial layers. MMP-2 was unchanged by l-NAME in the vascular smooth muscle. *P < 0.05 by Student's t-test; n = 6 LP; n = 5 LPLN.

Fig. 5.

Endothelial and adventitial TIMP-2 protein expression is decreased in late pregnant mUA isolated from lpln- rats. A: mUAs were dissected from LP and LPLN-treated animals, fixed, paraffin-embedded, cross-sectioned, and analyzed using immunofluorescence to detect TIMP-2 (green). DAPI was used to stain nuclei (blue). Images were taken at ×25 and ×100 as described in materials and methods. Bar = 50 μm. DAPI images represent the secondary antibody control images where there was no incubation with primary antibody. B: quantification of TIMP-2 protein immunofluorescence was measured using the integrated intensity function on MetaMorph software and normalizing for area measured. Relative integrated intensity is expressed as a ratio to LP control per area (μm²). TIMP-2 expression was significantly attenuated in the endothelial and adventitial layers of LPLN mUA. MUA TIMP-2 was unchanged by l-NAME in the vascular smooth muscle. *n = 7 LP; n = 7 LPLN, except n = 6 LP adventitia (Adv). C: LP and LPLN mUA were dissected and placed in modified RIPA buffer for protein analysis as described in materials and methods. Twenty-five micrograms of protein were loaded on the gel. Shown is a representative immunoblot. D: densitometry, as described in materials and methods, was used to quantify the immunoblots probed for TIMP-2 and β-tubulin. TIMP-2 is expressed as a ratio to β-tubulin. TIMP-2 is significantly decreased in late pregnant l-NAME-treated mUA. n = 6 LP; n = 5 LPLN. *P < 0.05 by Student's t-test.

Fig. 6.

Treatment of rats with l-NAME decreases MT1-MMP protein expression in LP mUA. Left: LP and LPLN mUA were dissected and placed in modified RIPA buffer for protein analysis as described in materials and methods. Twenty-five micrograms of protein were loaded on a denaturing gel. Shown is a representative immunoblot. Right: densitometry, as described in materials and methods, was used to quantify the immunoblots probed for MT1-MMP and β-tubulin. MT1-MMP is significantly decreased in mUA of LPLN animals compared with LP. *P < 0.05 by Student's t-test.