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1Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine and Magee-Women's Research Institute, and 3Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and 2Department of Zoology and Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia
Submitted 19 October 2005 ; accepted in final form 13 February 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-actin ratio = 0.29 vs. 0.21 (P < 0.01) and 0.43 vs. 0.32 (P < 0.005). Findings were similar for mesenteric arteries. MMP-2 mRNA as measured by real-time PCR was increased in small renal arteries from pregnant and Rlx-treated nonpregnant rats compared with their respective controls. There were no significant differences in tissue inhibitor of metalloproteinase (TIMP-1 or TIMP-2) activity by reverse zymography in small renal arteries. Thus increases in MMP-2 mRNA and protein expression are major factors contributing to increased MMP-2 activity in small arteries from pregnant and Rlx-treated nonpregnant rats. gelatinase; pregnancy; vasodilation
We recently showed that Rlx upregulates vascular gelatinase activity during pregnancy, thereby mediating renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries through activation of the endothelial ETB receptor-NO pathway (16). The hypothesis that matrix metalloproteinase-2 (MMP-2) plays a pivotal role in the endothelial ETB receptor-NO pathway was based on the confluence of several observations. 1) Rlx, the ETB receptor, and NO have an essential role in pregnancy-mediated renal vasodilation (see above). 2) Rlx was shown to upregulate MMP expression, at least in fibroblasts (23, 26, 27). 3) Vascular MMPs such as MMP-2 are able to process big ET at the Gly-Leu bond to ET1–32, with subsequent activation of ET receptors (9, 10).
An essential role for vascular gelatinase in the renal circulatory changes of pregnancy, which are mediated by Rlx (20), was demonstrated by inhibition of gelatinase activity in chronically instrumented rats in vivo and in isolated small renal arteries ex vivo. Moreover, vascular gelatinase, specifically, gelatinase A, or MMP-2 was upregulated by 
50% in small renal arteries harvested from Rlx-treated nonpregnant or midterm-pregnant rats. Thus vascular gelatinase activity is not only part of the endothelial ETB-NO vasodilatory pathway, it is also a major locus of regulation by Rlx. The lack of reduced myogenic reactivity of small renal arteries after Rlx administration to ETB receptor-deficient rats, in the face of an increase in MMP-2 activity, suggested that MMP-2 is in series with and upstream of the endothelial ETB-NO signaling pathway (16).
MMP-2 is secreted as a partially active proenzyme or zymogen (72 kDa) and is sequentially processed to an intermediate (64kDa) and then to an active (62kDa) form (1). The regulation of MMP-2 activity is complex, with possible loci of regulation at the level of transcription, translation, or posttranslational processing by endogenous activators and inhibitors, including tissue inhibitors of metalloproteinase (TIMPs). The regulation and function of MMP-2 are highly tissue specific. Moreover, arteries demonstrate significant heterogeneity among different organ beds, as well as within the same circulatory bed, depending on their location and size. Therefore, the goals of this study were 1) to further explore the mechanisms for the increased vascular MMP-2 activity elicited by pregnancy and Rlx in small renal arteries and 2) to determine whether these findings can be extended to other artery types.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Long-Evans female rats (12–14 wk old; Harlan Sprague Dawley, Frederick, MD) were fed PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds, St. Louis, MO), with water provided ad libitum, and maintained on a 12:12-h light-dark cycle. The Institutional Animal Care and Use Committee of the Magee-Women's Research Institute approved all animal procedures. Female rats destined to become pregnant were housed with a male breeder, and day 0 of pregnancy was documented by the presence of spermatozoa in the vaginal lavage. Virgin rats selected from the same shipment were used as controls. Other virgin female rats were implanted with an osmotic minipump containing recombinant human Rlx (rhRlx) or vehicle (20 mM sodium acetate, pH 5.0). The 7-day osmotic minipump (Alzet model 2001, Durect, Cupertino, CA) was inserted subcutaneously in the back of the animal under isoflurane anesthesia. It delivered rhRlx at the dose of 4 µg/h to yield concentrations of circulating Rlx similar to those measured on gestational days 10–12 (early to midterm) in pregnant rats, i.e., 14–20 ng/ml when pregnancy-induced renal vasodilation is maximal in this species (3, 8). All surgical procedures were conducted using aseptic technique. Pregnant rats were euthanized on gestational days 12–14 (midterm). Rlx- and vehicle-treated nonpregnant rats were euthanized on day 5 after implantation of the minipump and isolation of the vessels.
Isolation and protein preparation of mesenteric and small renal arteries.
A laparotomy was performed through the linea alba. For removal of the mesenteric arcade, we cut along the intestines, as close to the small bowel as possible to include the smallest vessels, with care taken to avoid entry into the bowel lumen. The tissue was immediately submerged and further dissected in ice-cold saline. A dissecting scope and jeweler's forceps were used to remove fat and connective tissue. The dissection was performed gently to minimize stretching of the vessels. The mesenteric cascade was then blotted on a gauze sponge, snap frozen, and stored at –80°C. Small (50- to 400-µm-ID) renal arteries were delicately dissected from the kidney (16, 17). The frozen small arteries from each rat were pooled, and protein homogenates were prepared as previously described in detail (16, 17). Protein concentrations were determined by a protein assay (Bio-Rad Laboratories, Hercules, CA), each in triplicate, and then averaged.
Evaluation of MMP-2 activity by gelatin zymography.
For gelatin zymography (16, 18), the homogenates were prediluted in Laemmli buffer (10 mM Tris, pH 6.8, 7 M urea, 10% glycerol, and 1% SDS) as needed, combined with an equal volume of Novex Tris-glycine SDS sample buffer (Invitrogen, Carlsbad, CA), and then allowed to stand at room temperature for 10 min. For small renal and mesenteric arteries, 7.5 and 5 µg of protein, respectively, were loaded in each lane and then electrophoresed on Novex precast 10% Tris-glycine gels containing 0.1% gelatin for 2 h at 100 V. After renaturation and equilibration, the buffer was replaced with fresh Novex zymogram developing buffer, and the gels were incubated at 37°C for various lengths of time depending on the experiment (generally 
18 h). Incubation time was optimized to avoid overdevelopment and consequent saturation of band densities. Gels were stained with 100 ml of fresh 0.5% Coomassie blue G250 in 30% methanol-10% acetic acid for 70 min and rinsed twice for 15 min each in a destaining solution of 30% methanol-10% acetic acid. The gels were further destained for 1 h in 1% Triton X-100 solution and then stored in distilled water until scanning densitometry was performed. The gels were scanned using a Scan Jet 5370C scanner (Hewlett-Packard, Palo Alto, CA) and PrecisionScan Pro version 1.4 computer program (Hewlett-Packard). The images were digitized for analysis by the UN-SCAN-IT gel automated digitizing system (version 4.3, Silk Scientific, Orem, UT). Bands of interest were delineated, and relative densitometries were calculated on the basis of the number of pixels. To combine the results from several zymograms and avoid interassay variability, the densitometric ratios of matched pairs of rhRlx-vehicle and pregnant-virgin were calculated and averaged for presentation.
Evaluation of MMP-2 protein by immunoblotting techniques.
MMP-2 protein was evaluated using Western blotting techniques. Tissue homogenates prepared as described above were diluted in Laemmli buffer according to Bio-Rad protein estimations before combination with an equal volume of loading buffer. For the small renal and mesenteric arteries, 50 and 10 µg of protein, respectively, were loaded per lane. Protein samples were electrophoresed on 7.5% SDS-polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked in nonfat dry milk (50 mg/ml Tris-buffered saline with 0.05% Tween) for 1 h at room temperature. They were incubated with a mouse monoclonal antibody for MMP-2 (EMD Biosciences, San Diego, CA) or mouse IgG1
(Sigma-Aldrich, St. Louis, MO) diluted in nonfat dry milk (both at a final concentration 1.6 µg/ml) overnight at 4°C. This MMP-2 antibody is directed against an epitope at the NH2 terminus corresponding to amino acids 468–483 of human MMP-2 (which is present in the pro- and active forms of MMP-2) with species reactivity to human, rat, and mouse. According to the manufacturer, this MMP-2 antibody recognizes both pro- and active forms of the MMP-2 protein. A mouse monoclonal antibody for -actin (Sigma, St. Louis, MO) was added at a concentration of 0.02 µg/ml, and the membranes were incubated for 1 h at room temperature. The membranes were washed with Tris-buffered saline with 0.05% Tween and then incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG that had been rat adsorbed (0.23 µg/ml; Cederlane Laboratories, Hornby, ON, Canada) for 1 h at room temperature. Chemiluminescence techniques were used for detection as previously described (17). Scanning densitometry was performed using PrecisionScan Pro version 1.4 software to quantify protein expression. MMP-2 protein expression was normalized to -actin for each sample.
Reverse zymography.
For reverse zymography (22), the prepared homogenates were diluted as needed in Laemmli buffer and combined with an equal volume of Novex Tris-glycine SDS sample buffer (Invitrogen). For the small renal and mesenteric arteries, 10 and 2.5 µg of protein, respectively, were loaded per lane. TIMP-1 and TIMP-2 standards were loaded on each gel (EMD Biosciences). Protein samples were electrophoresed at 75 V for 2.5 h on a standard separating gel composed of 2.25 mg/ml porcine gelatin, 0.25 M Tris·HCl (pH 8.8), 0.125% SDS, 1 µl/ml N,N,N',N'-tetramethylethylenediamine, 0.4 mg/ml ammonium persulfate, 15% acrylamide, 0.4% bisacrylamide, and 100 ng/ml proenzyme MMP-2 (EMD Biosciences). A 4% stacking gel was used. After electrophoresis, the gels were incubated in 1x Novex zymogram renaturing buffer with gentle agitation at room temperature for 3 h, with replacement of the solution at 1-h intervals. Then the renaturing buffer was replaced with 100 ml of 1x Novex developing buffer. The gel was then incubated at 37°C overnight for 15 h. Each gel was stained with 0.5% Coomassie blue G250 in 30% methanol-10% acetic acid for 4 h and then washed four times with destaining solution containing 30% methanol-10% acetic acid for 1, 15, 30, and 60 min. The gels were further destained in 1% Triton X-100 solution for 1 h and stored in distilled water until densitometry was performed. To combine the results from several gels and avoid interassay variability, the densitometric ratios of matched pairs of rhRlx-vehicle and pregnant-virgin were calculated and averaged for presentation.
Immunohistochemistry.
Kidney sections from rats were also prepared for immunohistochemistry to examine blood vessels in situ. The kidneys were harvested and stripped of the capsule and perihilar fat. The poles of the kidney were removed at both ends. The remaining kidney tissue was bisected at the hilum across the short axis, thus yielding two pieces. For frozen sections, the kidney pieces were oriented in cryomolds (Sakura Finetek, Torrance, CA) in such a way as to provide a complete cross section of the kidney when sectioned and mounted onto slides. Individual arteries were isolated from the mesenteric arcade and similarly prepared. Frozen 7-µm-thick sections were cut by cryostat, mounted on slides, fixed in acetone for 10 min, and washed twice for 3 min each in PBS. Slides were allowed to dry at room temperature and stored at –40°C until use.
After a dose response (0.1–10 µg/ml) was investigated, an optimal dose of 3 µg/ml was used for the same MMP-2 mouse monoclonal antibody described above (see Evaluation of MMP-2 protein by immunoblotting techniques). For a negative control, the same concentration of mouse IgG1 was substituted for the primary antibody. The primary and negative control antibodies were preabsorbed overnight with 10% rat serum to increase specificity.
After permeabilization with 0.3% Triton X-100, quenching of endogenous peroxidase with 1% hydrogen peroxide in methanol, and blocking with normal horse serum, the sections were incubated with the primary antibody or its negative control overnight at 4°C. After the slides were washed in PBS, sections were incubated for 30 min with a biotinylated, rat-adsorbed secondary antibody against mouse IgG (Vector Laboratories, Burlingame, CA). The Vectastain Elite ABC kit was used to detect the immunoreactivity of MMP-2 with diaminobenzidine as the chromogen substrate. Mesenteric artery sections were counterstained with hematoxylin. After dehydration in ethanol and xylene solutions, a coverslip was applied using Cytoseal XYL (Stephens Scientific, Riverdale, NJ).
Real-time PCR.
Real-time PCR was used for relative quantification of MMP-2 gene expression in small renal arteries from Rlx- and vehicle-treated rats and from midterm-pregnant and virgin rats (25). Total RNA was isolated using RNAwiz (Ambion, Austin, TX) and resuspended in water treated with RNA Secure (Ambion). Gel electrophoresis confirmed the quality of the isolated total RNA from each sample. Total RNA from small renal arteries of six midterm-pregnant and six virgin rats and six Rlx- and six vehicle-treated nonpregnant rats was combined into two pools of three samples each. For each pooled sample, 2.0 µg of total RNA were reverse transcribed in a 40-µl reaction containing 1x RT buffer, 5.5 mM MgCl2, 2 mM deoxynucleotide triphosphates, 2.5 µM random hexamers, 1.25 U of Multiscribe RT, and 1 U of RNase inhibitor (all Applied Biosystems, Foster City, CA). In separate reactions, 30 ng of total RNA per pooled sample were reversed transcribed for PCR of the 18S ribosomal RNA endogenous reference gene. cDNA synthesis of all samples was carried out simultaneously at 42°C for 45 min.
PCR primers and TaqMan probes for the detection of MMP-2 and 18S mRNA were designed using the Primer Express software program (Applied Biosystems). The rat specific MMP-2 forward and reverse primers were designed in a region of the sequence that shared low homology with other MMP sequences. The specific target was a 75-bp fragment from nucleotides 1579–1654 in exon 8 of the rat MMP-2 mRNA sequence. The sequences of the primers were as follows: 5'-ACGATGGCAAGGTGTGGTGT-3' (forward) and 5'-CCTTGGTCAGGACAGAAGCC-3' (reverse) for MMP-2 and 5'-CGGCTACCACATCCAAGGAA-3' (forward) and 5'-GCTGGAATTACCGCGGCT-3' (reverse) for 18S. 6-Carboxyfluorescein-labeled probes were also used: 5'-6FAM-ACCACAACCAACTACGATGATGACCGGA-TAMRA-3' for MMP-2 and 5'-6FAM-TGCTGGCACCAGACTTGCCCTC-TAMRA-3' for 18S.
Real-time PCR was performed in an ABI PRISM 7700 sequence detector using 96-well optical plates (Applied Biosystems). Each well contained a total volume of 50 µl, which consisted of 5 µl of cDNA template, 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 0.8 µM forward and reverse primers, and 0.4 µM probe. Relative quantification of MMP-2 gene expression was performed using the comparative cycle threshold (Ct) method as described in ABI User Bulletin 2, which consists of normalization of the number of target gene copies (MMP-2) to an endogenous reference (18S) and relative to a calibrator (virgin rats). Each sample was tested in triplicate, and an average Ct value was calculated for the replicates. Relative quantification of MMP-2 gene expression was performed comparing small renal arteries from pregnant rats, Rlx-treated nonpregnant rats, and vehicle-treated nonpregnant rats with gene expression in small renal arteries from virgin rats (calibrator) using the following formula: 2
, where 
Ct = average MMP-2 Ct – average 18S Ct and CT = Ct – Ct (virgin) (Table 1).
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Values are means ± SE. All data were found to be normally distributed. Therefore, parametric tests were utilized. For MMP-2 and TIMP activities, the densitometric ratio for each matched pair of pregnant and virgin, as well as rhRlx- and vehicle-treated rats was calculated. Average ratios were then determined, and Student's t-test was applied comparing these ratios with 1.0. A ratio of 1.0 would indicate no difference between the groups. For MMP-2 protein as determined by immunoblotting, pro-MMP-2 protein was normalized to -actin. Pregnant and Rlx-treated nonpregnant groups were compared with their respective controls by t-test. Because increases in gelatinase activity and protein ratios were expected to be >1.0 in small arteries from midterm-pregnant and rhRlx-treated nonpregnant rats (16), P values for one tail are presented. For analysis of TIMPs, a two-tailed t-test was utilized.
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RESULTS |
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The pattern of MMP-2 activity was similar in the mesenteric arteries (Fig. 1). In these vessels, MMP-2 activity was upregulated in pregnant compared with virgin vessels (pro-MMP-2 by 50% and active MMP-2 by 40%, both P < 0.05). Comparable increases were seen in mesenteric arteries from Rlx vs. vehicle-treated nonpregnant rats (pro-MMP-2 by 50% and active MMP-2 by 90%, both P < 0.005). For the latter, the increase in active MMP-2 exceeded that of pro-MMP-2 (P < 0.05). We also demonstrated an increase in pro- and active MMP-2 activities in thoracic aortae from pregnant compared with virgin rats (ratios = 2.2 ± 0.5 and 1.4 ± 0.2, respectively, P < 0.05, n = 10 pairs), as well as Rlx-treated compared with vehicle-treated rats (ratios = 1.4 ± 0.2 and 1.7 ± 0.2, respectively, P < 0.05, n = 10 pairs).
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-actin are depicted in Fig. 2. Pro-MMP-2 was increased in small renal arteries from pregnant compared with virgin rats (pro-MMP-2-to--actin ratio = 0.29 ± 0.02 vs. 0.21 ± 0.02, P < 0.01). Pro-MMP-2 protein was increased in small renal arteries from nine of nine matched pregnant-virgin pairs (mean ratio = 1.52 ± 0.21), thereby indicating an overall 52% increase in pro-MMP-2 protein in arteries from the gravid rats. Pro-MMP-2 protein was also increased in small renal arteries from Rlx- compared with vehicle-treated control rats (0.42 ± 0.02 vs. 0.32 ± 0.02, P < 0.005). This ratio was >1.0 in 10 of 11 matched Rlx-vehicle pairs (mean ratio of pro-MMP-2 among matched pairs of 1.36 ± 0.09, P < 0.005). Active MMP-2 protein expression was too low to be accurately quantitated by imaging (see DISCUSSION). Mesenteric arteries from pregnant and Rlx-treated nonpregnant rats also showed an increase in pro-MMP-2 protein expression (Fig. 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Major results were as follows. Small renal arteries from midterm-pregnant and Rlx-treated nonpregnant rats demonstrated increased pro- and active MMP-2 activities by gelatin zymography, increased pro-MMP-2 protein by Western analysis, and increased MMP-2 mRNA expression by real-time PCR. Comparable findings were observed in another arterial bed, namely, mesenteric arteries from midterm-pregnant and Rlx-treated nonpregnant rats (MMP-2 mRNA was not investigated in these arteries). TIMP-1 and TIMP-2 activities were not altered, except in small renal and mesenteric arteries from the Rlx-treated rats, where TIMP-2 tended to be increased or was significantly increased, respectively. Finally, MMP-2 protein expression was associated with both the endothelium and vascular smooth muscle in the small renal and mesenteric arteries.
The regulation and function of MMPs are complex (1, 5, 19). MMP-2 is secreted as a proenzyme or zymogen (72 kDa), which is sequentially processed to intermediate (64kDa) and active (62kDa) forms. TIMP-2 has a dual role in MMP-2 regulation depending on its abundance. Low to moderate levels contribute to pro-MMP-2 activation by tethering pro-MMP-2 to membrane type 1 (MT1)-MMP, which is associated with the cell membrane. A neighboring MT1-MMP then catalyzes the formation of active MMP-2 from the tethered zymogen. High levels of TIMP-2, however, preempt and inhibit all membrane-associated MT1-MMP, thereby precluding activation of pro-MMP-2. In addition, TIMP-2 can directly inhibit active MMP-2 by interacting with the catalytic site.
Our findings of elevated pro- and active MMP-2 activities in small renal arteries from midterm-pregnant and Rlx-treated nonpregnant rats by gelatin zymography corroborate our earlier investigation (16). Here, we also document comparable findings for mesenteric arteries. We also extend this finding of increased pro- and active MMP-2 activities to large vessels, specifically, thoracic aortae from pregnant rats and Rlx-treated nonpregnant rats compared with their respective controls. The increase in pro- and active MMP-2 was approximately equal in small renal and mesenteric arteries isolated from the midterm-pregnant rats by gelatin zymography. However, in both artery types isolated from Rlx-treated rats, the increase in active MMP-2 activity was significantly greater than for pro-MMP-2 by zymography. [There was a similar trend in our previous investigation (16).] This finding may be due to the increase in TIMP-2 activity expressed by the arteries isolated from Rlx-treated rats, which reached significance for the mesenteric vessels (Fig. 5, B and D). As described above, modest increases in TIMP-2 activity facilitate pro-MMP-2 processing to the active form. Thus Rlx administration to nonpregnant rats may not completely mimic the pregnant condition.
The elevation in pro-MMP-2 activity observed on gelatin zymography in both artery types stimulated by pregnancy or Rlx was corroborated by an increase in pro-MMP-2 protein expression as determined by Western analysis. Pro-MMP-2 activity is not biologically relevant but, rather, a by-product of gelatin zymography. Moreover, even though the antibody recognizes pro- and active MMP-2, we were unable to corroborate the concurrent increase in active MMP-2 observed on gelatin zymography by Western analysis. This is most likely because, on a molar basis, active MMP-2 is three times more active on zymography than pro-MMP-2 (29). In other words, the active MMP-2 protein expression was below the sensitivity of reliable detection by Western analysis. Rlx has been shown to downregulate TIMPs, specifically TIMP-1 expression, in human dermal and lower uterine segment fibroblasts (23, 26). However, we did not observe downregulation by Rlx (or pregnancy) of TIMP-1 or TIMP-2 activity in isolated small renal or mesenteric arteries. Thus this mechanism is unlikely to contribute to the increase in the active MMP-2 activity (i.e., by reducing the level of an inhibitor). However, we cannot rule out the potential contribution of other inhibitors such as 
2-macroglobulin (12) or "reversion-inducing cysteine-rich protein with kazal motifs" (RECK) (28), which might be downregulated by pregnancy or Rlx. Nor did we evaluate MT1-MMP (30), which could potentiate pro-MMP-2 activation, especially in the Rlx-treated rats (perhaps in concert with increases in TIMP-2 as discussed above), or extracellular MMP inhibitor (EMMPRIN) (11, 15), which can increase MT1-MMP and pro-MMP-2 protein expression.
Nevertheless, given the increase in pro-MMP-2 protein expression, we further tested whether augmented gene transcription contributes. We investigated MMP-2 mRNA expression by real-time RT-PCR in small renal arteries pooled from three midterm-pregnant and three virgin rats, as well as from three Rlx- and three vehicle-treated nonpregnant rats. Relative MMP-2 mRNA expression was increased in the arteries from the pregnant and Rlx-treated nonpregnant rats. This finding was duplicated using a second pool of small renal arteries from four additional groups of animals (Table 1).
We found MMP-2 immunoreactivity to be associated with the vascular smooth muscle and endothelium in the small renal and mesenteric arteries. Because immunohistochemistry is only semiquantitative and unlikely to have the sensitivity to detect 30–40% differences in protein expression as revealed by our Western analysis, we were unable to determine whether the increase induced by pregnancy or Rlx occurred in the vascular smooth muscle and/or endothelium. There is evidence that, in the endothelial cell, MMP-2 and its associated proteins MT1-MMP and TIMP-2, as well as the ETB receptor and endothelial NO synthase, are localized to caveolae (2).
In summary, the present results indicate that the increase in active MMP-2 in the vascular wall induced by pregnancy or Rlx administration to nonpregnant animals occurs because of increased pro-MMP-2 expression, the latter as a consequence, wholly or in part, of increased pro-MMP-2 mRNA. Increased TIMP-2 may further contribute to elevated MMP-2 activity in the vasculature of Rlx-treated nonpregnant animals. Moreover, these biochemical changes occur not only in small renal arteries, but also in other types of arteries, i.e., small mesenteric arteries and aortae. Whether human arteries are similarly affected by pregnancy or Rlx requires additional study. Further delineation of the molecular mechanisms by which pregnancy or Rlx augments MMP-2 mRNA in the vascular wall and localization of the vascular site(s) of increase remain to be explored.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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expression, but not relaxin receptors (LGR7), in the myometrium of late pregnant relaxin gene knockout mice. Endocrinology 144: 4272–4275, 2003.This article has been cited by other articles:
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P < 0.05 vs. pro-MMP-2.
